Novel anti-pd-1 antibodies

ABSTRACT

The present disclosure provides monoclonal antibodies against protein programmed cell death 1 (PD-1), which can block the binding of PD-1 ligands to PD-1, and therefore block the inhibitory function of PD-1 ligands on PD-1 expressing T cells. The antibodies of disclosure provide very potent agents for the treatment of multiple cancers via modulating human immune function.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/239,866, filed Apr. 26, 2021, which is a divisional of U.S. patentapplication Ser. No. 15/751,236, filed Feb. 8, 2018, now U.S. Pat. No.11,008,391, which is a national stage entry, filed under 35 U.S.C. §371, of International Application No. PCT/CN2016/094624, filed Aug. 11,2016, which claims the benefit of priority to International ApplicationNo. PCT/CN2016/071374, filed Jan. 19, 2016, and InternationalApplication No. PCT/CN2015/086594, filed Aug. 11, 2015, the contents ofall of which are incorporated herein by reference in their entireties.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (37JD-337840-US3.xml;Size: 66,233 bytes; and Date of Creation: Mar. 18, 2023) is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure generally relates to novel anti-PD-1 antibodies.

BACKGROUND

Increasing evidences from preclinical and clinical results have shownthat targeting immune checkpoints is becoming the most promisingapproach to treat patients with cancers. Programmed cell death 1, one ofimmune-checkpoint proteins, play a major role in limiting the activityof T cells that provide a major immune resistance mechanism by whichtumor cells escaped immune surveillance. The interaction of PD-1expressed on activated T cells, and PD-L1 expressed on tumor cellsnegatively regulate immune response and damp anti-tumor immunity.Expression of PD-L1 on tumors is correlated with reduced survival inesophageal, pancreatic and other types of cancers, highlighting thispathway as a new promising target for tumor immunotherapy. Multipleagents targeting PD-1 pathway have been developed by pharmaceuticalcompanies, such as Bristol-Myers Squibb (BMS), Merck, Roche andGlaxoSmithKline (GSK). Data from clinical trials demonstrated earlyevidence of durable clinical activity and an encouraging safety profilein patients with various tumor types. Nivolumab, a PD-1 drug developedby BMS, is being put at center stage of the next-generation field. Nowin 6 late-stage studies, the treatment spurred tumor shrinkage in threeof 5 cancer groups studied, including 18% of 72 lung cancer patients,close to a third of 98 melanoma patients and 27% of 33 patients withkidney cancer. Developed by Merck, lambrolizumab is a fully humanmonoclonal IgG4 antibody that acts against PD-1, which grabbed the FDA'snew breakthrough designation after impressive IB data came through forskin cancer. The results from a phase IB study have shown an objectiveanti-tumor response in 51% of 85 cancer patients, and a completeresponse in 9% of patients. Roche's experimental MPDL3280A demonstratedan ability to shrink tumors in 29 of 140(21%) advanced cancer patientswith various tumor sizes.

However, the existing therapies may not be all satisfactory andtherefore new anti-PD-1 antibodies are still needed.

BRIEF SUMMARY OF THE INVENTION

The present disclosure provides novel monoclonal anti-PD-1 antibodies(in particular fully human antibodies), polynucleotides encoding thesame, and methods of using the same.

In one aspect, the present disclosure provides isolated monoclonalantibodies or antigen binding fragments thereof, which are capable ofspecifically binding to human PD-1 at a Kd value no more than 10⁻⁸ M(e.g. no more than ≤9×10⁻⁹ M, ≤8×10⁻⁹ M, ≤7×10⁻⁹ M, ≤6×10⁻⁹ M, ≤5×10⁻⁹M, ≤4×10⁻⁹ M, ≤3×10⁻⁹ M, ≤2×10⁻⁹ M, or ≤10⁻⁹ M) as measured by plasmonresonance binding assay.

In certain embodiments, the antibodies or antigen binding fragmentsthereof bind to monkey PD-1 at an EC50 of no more than 100 nM or no morethan 10 nM (e.g. no more than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM). In certainembodiments, the antibodies and antigen-binding fragments thereof do notbind to mouse PD-1 but bind to monkey PD-1 with a binding affinitysimilar to that of human PD-1. In certain embodiments, the antibodies orantigen binding fragments thereof potently inhibit binding of human ormonkey PD-1 to its ligand (e.g. PD-L1 or PD-L2), at an IC50 of no morethan 100 nM (e.g. no more than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM,8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM,0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM). In certainembodiments, the EC50 or IC50 is measured by fluorescence-activated cellsorting (FACS) analysis.

In certain embodiments, the antibodies or antigen binding fragmentsthereof have substantially reduced effector function. In certainembodiments, the antibodies or antigen binding fragments thereof do notmediate ADCC or CDC or both.

In certain embodiments, the antibodies or antigen binding fragmentsthereof provided herein comprise a heavy chain CDR sequences selectedfrom the group consisting of: SEQ ID NOs: 1, 3, 5, 13, 15, 21, 23, 25,33, 35 and 37.

In one aspect, the antibodies or an antigen binding fragments thereofprovided herein comprise a light chain CDR sequences selected from thegroup consisting of: SEQ ID NOs: 7, 9, 11, 17, 19, 27, 29, 31, 39, 41,43 and 65.

In certain embodiments, the antibodies or antigen binding fragmentsthereof provided herein comprise at least one, two, three, four, five orsix CDRs selected from the group consisting of: SEQ ID NOs: 1, 3, 5, 7,9, and 11; or selected from the group consisting of: SEQ ID NOs: 13, 15,5, 7, 17 and 11; or selected from the group consisting of: SEQ ID NOs:1, 15, 5, 7, 17 and 19; or selected from the group consisting of: SEQ IDNOs: 1, 15, 5, 7, 17, and 65; or selected from the group consisting of:SEQ ID NOs: 21, 23, 25, 27, 29 and 31; or selected from the groupconsisting of: SEQ ID NOs: 33, 35, 37, 39, 41 and 43.

In certain embodiments, the antibodies or antigen binding fragmentsthereof provided herein comprise a heavy chain variable region selectedfrom the group consisting of:

-   -   a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID        NO: 3, and/or SEQ ID NO: 5;    -   b) a heavy chain variable region comprising SEQ ID NO: 13, SEQ        ID NO: 15, and/or SEQ ID NO: 5;    -   c) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID        NO: 15, and/or SEQ ID NO: 5;    -   d) a heavy chain variable region comprising SEQ ID NO: 21, SEQ        ID NO: 23, and/or SEQ ID NO: 25; and    -   e) a heavy chain variable region comprising SEQ ID NO: 33, SEQ        ID NO: 35, and/or SEQ ID NO: 37.

In certain embodiments, the antibodies or antigen binding fragmentsthereof provided herein comprise a light chain variable region selectedfrom the group consisting of:

-   -   a) a light chain variable region comprising SEQ ID NO: 7, SEQ ID        NO: 9, and/or SEQ ID NO: 11;    -   b) a light chain variable region comprising SEQ ID NO: 7, SEQ ID        NO: 17, and/or SEQ ID NO: 11;    -   c) a light chain variable region comprising SEQ ID NO: 7, SEQ ID        NO: 17, and/or SEQ ID NO: 19;    -   d) a light chain variable region comprising SEQ ID NO: 27, SEQ        ID NO: 29, and/or SEQ ID NO: 31;    -   e) a light chain variable region comprising SEQ ID NO: 39, SEQ        ID NO: 41, and/or SEQ ID NO: 43; And    -   f) a light chain variable region comprising SEQ ID NO: 7, SEQ ID        NO: 17, and/or SEQ ID NO: 65.

In certain embodiments, the antibodies or antigen binding fragmentsthereof provided herein comprise:

-   -   a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID        NO: 3, and/or SEQ ID NO: 5; and a light chain variable region        comprising SEQ ID NO: 7, SEQ ID NO: 9, and/or SEQ ID NO: 11;    -   b) a heavy chain variable region comprising SEQ ID NO: 13, SEQ        ID NO: 15, and/or SEQ ID NO: 5; and a light chain variable        region comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO:        11;    -   c) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID        NO: 15, and/or SEQ ID NO: 5; and a light chain variable region        comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 19;    -   d) a heavy chain variable region comprising SEQ ID NO: 21, SEQ        ID NO: 23, and/or SEQ ID NO: 25 and a light chain variable        region comprising SEQ ID NO: 27, SEQ ID NO: 29, and/or SEQ ID        NO: 31;    -   e) a heavy chain variable region comprising SEQ ID NO: 33, SEQ        ID NO: 35, and/or SEQ ID NO: 37; and a light chain variable        region comprising SEQ ID NO: 39, SEQ ID NO: 41, and/or SEQ ID        NO: 43; or    -   f) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID        NO: 15, and/or SEQ ID NO: 5; and a light chain variable region        comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 65.

In certain embodiments, the antibodies or antigen binding fragmentsthereof provided herein comprise a heavy chain variable region selectedfrom the group consisting of: SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO:53, SEQ ID NO: 57 and SEQ ID NO: 61.

In certain embodiments, the antibodies or antigen binding fragmentsprovided herein comprise a light chain variable region selected from thegroup consisting of: SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ IDNO: 59, SEQ ID NO: 63 and SEQ ID NO: 67.

In certain embodiments, the antibodies or antigen binding fragmentsthereof provided herein comprise:

-   -   a) a heavy chain variable region comprising SEQ ID NO: 45; and a        light chain variable region comprising SEQ ID NO: 47;    -   b) a heavy chain variable region comprising SEQ ID NO: 49; and a        light chain variable region comprising SEQ ID NO: 51;    -   c) a heavy chain variable region comprising SEQ ID NO: 53; and a        light chain variable region comprising SEQ ID NO: 55;    -   d) a heavy chain variable region comprising SEQ ID NO: 57; and a        light chain variable region comprising SEQ ID NO: 59;    -   e) a heavy chain variable region comprising SEQ ID NO: 61; and a        light chain variable region comprising SEQ ID NO: 63; or    -   f) a heavy chain variable region comprising SEQ ID NO: 53; and a        light chain variable region comprising SEQ ID NO: 67.

In certain embodiments, the antibodies provided herein include, forexample, 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.103.11-v2 hAb, 1.139.15hAb, and 1.153.7 hAb.

In certain embodiments, the antibodies or antigen binding fragmentsthereof provided herein compete for the same epitope with antibodies1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.103.11-v2 hAb, 1.139.15 hAb, or1.153.7 hAb. In certain embodiments, the antibodies or antigen bindingfragments thereof provided herein bind to the epitope comprising atleast one of the following amino acid residues of PD-1:V64, P83, D85,L128, A129, P130, K131, A132 and Q133.

In certain embodiments, the antibodies or antigen binding fragmentsthereof are capable of blocking binding of human PD-1 to its ligand andthereby providing at least one of the following activities:

-   -   a) inducing production of IL-2 in CD4+T cells;    -   b) inducing production of IFNγ in CD4+T cells;    -   c) inducing proliferation of CD4+T cells and    -   d) reversing T reg's suppressive function.

In certain embodiments, the antibodies provided herein are a monoclonalantibody, fully human antibody, humanized antibody, chimeric antibody,recombinant antibody, bispecific antibody, labeled antibody, bivalentantibody, or anti-idiotypic antibody. In certain embodiments, theantibodies or antigen binding fragments thereof are fully humanmonoclonal antibodies, optionally produced by a transgenic rat, forexample, a transgenic rat having inactivated endogenous expression ofrat immunoglobulin genes and carrying recombinant human immunoglobulinloci having J-locu deletion and a C-kappa mutation.

In certain embodiments, the antigen-binding fragments thereof providedherein are a camelized single domain antibody, a diabody, a scFv, anscFv dimer, a BsFv, a dsFv, a (dsFv)2, a dsFv-dsFv′, an Fv fragment, aFab, a Fab′, a F(ab′)2, a ds diabody, a nanobody, a domain antibody, ora bivalent domain antibody.

In certain embodiments, the antibodies or antigen-binding fragmentsthereof provided herein further comprise an immunoglobulin constantregion.

In certain embodiments, the antibodies or antigen-binding fragmentsthereof provided herein, further comprise a conjugate.

In certain embodiments, the conjugate can be a detectable label, apharmacokinetic modifying moiety, or a purification moiety.

In another aspect, the present disclosure provides isolatedpolynucleotides encoding the antibodies or antigen binding fragmentsthereof provided herein. In certain embodiments, polynucleotides areprovided that encode the amino acid sequences of the antibodies orantigen-binding fragments disclosed herein. In certain otherembodiments, vectors are provided that comprise these polynucleotides,and in certain other embodiments, host cells are provided that comprisethese vectors. In certain embodiments, methods are provided forexpressing one or more of the antibodies or antigen-binding fragmentsdisclosed herein by culturing these host cells under conditions in whichthe antibodies or antigen-binding fragments encoded by thepolynucleotides are expressed from a vector. In certain embodiments, thepolynucleotides provided herein are operably associated with a promotersuch as a SV40 promoter in a vector. In certain embodiments, host cellscomprising the vectors provided herein are Chinese hamster ovary cell,or 293F cell.

In another aspect, the present disclosure provides kits comprising theantibody or antigen-binding fragment thereof.

In another aspect, the PD-1 antibodies provided herein, such as the1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.103.11-v2 hAb, 1.139.15 hAb, and1.153.7 hAb have good tolerability and high in vivo anti-tumor activityin an animal. In certain embodiments, an animal having tumor cellsadministered with the PD-1 antibodies provided herein has a reduction ofthe tumor volume by at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least95% as compared to the control animal having similar baseline tumorvolume but administered only with vehicle.

In another aspect, the present disclosure provides methods of treating acondition associated with PD-1 in an individual, comprising:administering to the individual a therapeutically effective amount ofantibody or antigen-binding fragment thereof provided herein. In certainembodiments, the individual has been identified as having a disorder ora condition likely to respond to a PD-1 antagonist. In certainembodiments, the individual has been identified as positive for presenceor upregulated level of the PD-L1 in a test biological sample from theindividual.

In another aspect, the present disclosure provides pharmaceuticalcompositions comprising the antibody or antigen-binding fragment thereofprovided herein and one or more pharmaceutically acceptable carriers. Incertain of these embodiments, the pharmaceutical carriers may be, forexample, diluents, antioxidants, adjuvants, excipients, or non-toxicauxiliary substances.

In another aspect, the present disclosure provides methods of treating acondition in a subject that would benefit from upregulation of immuneresponse, comprising administering an effective amount of the antibodyor antigen-binding fragment thereof provided herein to the subject. Incertain embodiments, the subject has upregulated expression of PD-L1, orhas been identified as positive for expression of PD-L1.

Use of the antibody or antigen-binding fragment thereof provided hereinin the manufacture of a medicament for treating a condition that wouldbenefit from upregulation of immune response. In certain embodiments,the condition is cancer or chronic viral infection.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 presents the binding of fully human anti-PD-1 antibodies to PD-1expressing CHO cell as measured by FACS analysis.

FIG. 2 presents the binding of fully human PD-1 antibodies to PD-1expressing CHO cell with EC50 about 2 nM as measured by FACS analysis.

FIG. 3 is the binding of fully human anti-PD-1 antibody to PD-1expressed on activated CD4+T cell as measured by FACS analysis.

FIG. 4 shows that the fully human anti-PD-1 antibodies blocked thebinding of PD-L1 to PD-1 transfected CHO cells with IC50 of about 3-8 nMas measured by FACS analysis.

FIG. 5 shows that the fully human anti-PD-1 antibodies specifically bindto PD-1, but do not bind family members CD28 and CTLA4, as measured byFACS analysis.

FIG. 6 shows that the fully human anti-PD-1 antibodies against PD-1 bindto cynomolgus monkey PD-1 but not murine PD-1.

FIG. 7 is the full kinetics of binding affinity of PD-1 antibodies tohuman PD-1 ranging from 3.76E-9 to 1.76E-10 mol/L as determined bysurface plasmon resonance.

FIG. 8 illustrates the effect of fully human anti-PD-1 antibodies onIL-2 production in mixed lymphocyte reaction (MLR).

FIG. 9 illustrates the effect of fully human anti-PD-1 antibodies onIFNγ production in MLR.

FIG. 10 shows that fully human anti-PD-1 antibodies promoted T cellproliferation in MLR.

FIG. 11 shows that fully human PD-1 antibodies promoted T cellproliferation in specific T cell response.

FIG. 12 shows that anti-PD-1 antibodies reversed Treg's suppressivefunction.

FIG. 13 shows that the anti-PD-1 antibodies lacked ADCC on activated Tcells.

FIG. 14 shows that the anti-PD-1 antibodies lacked CDC on activated Tcells.

FIG. 15 shows that 1.103.11-v2 hAbs in different buffers bind to humanPD-1 extracellular domain with similar affinity measured by ELISA.“1.103.11-v2 hAb in buffer” refers to the antibody in the formulationbuffer, and “1.103.11-v2 hAb in PBS” refers to antibody in the 1×PBS, pH7.4.

FIG. 16 shows that 1.103.11-v2 hAbs in different buffers bind to PD-1expressing CHO cell with similar affinity measured by FACS. “1.103.11-v2hAb in buffer” refers to the antibody in the formulation buffer, and“1.103.11-v2 hAb in PBS” refers to antibody in the 1×PBS, pH 7.4.

FIG. 17 shows the hot-spot residues (shadow area) on the crystalstructure of the human PD-L1 that antibodies bind to. A shows the commonhot-spot residues; B-D show the hot-spot residues for 1.103.11 hAb,Keytruda and 11.148.10 hAb, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The following description of the disclosure is merely intended toillustrate various embodiments of the disclosure. As such, the specificmodifications discussed are not to be construed as limitations on thescope of the disclosure. It will be apparent to one skilled in the artthat various equivalents, changes, and modifications may be made withoutdeparting from the scope of the disclosure, and it is understood thatsuch equivalent embodiments are to be included herein. All referencescited herein, including publications, patents and patent applicationsare incorporated herein by reference in their entirety.

Definitions

The term “antibody” as used herein includes any immunoglobulin,monoclonal antibody, polyclonal antibody, multispecific antibody, orbispecific (bivalent) antibody that binds to a specific antigen. Anative intact antibody comprises two heavy chains and two light chains.Each heavy chain consists of a variable region and a first, second, andthird constant region, while each light chain consists of a variableregion and a constant region. Mammalian heavy chains are classified asα, δ, ε, γ, and μ, and mammalian light chains are classified as λ or κ.The antibody has a “Y” shape, with the stem of the Y consisting of thesecond and third constant regions of two heavy chains bound together viadisulfide bonding. Each arm of the Y includes the variable region andfirst constant region of a single heavy chain bound to the variable andconstant regions of a single light chain. The variable regions of thelight and heavy chains are responsible for antigen binding. Thevariables region in both chains generally contain three highly variableloops called the complementarity determining regions (CDRs) (light (L)chain CDRs including LCDR1, LCDR2, and LCDR3, heavy (H) chain CDRsincluding HCDR1, HCDR2, HCDR3). CDR boundaries for the antibodies andantigen-binding fragments disclosed herein may be defined or identifiedby the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B.,Chothia, C., Lesk, A. M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C.et al., J Mol Biol. December 5; 186(3):651-63 (1985); Chothia, C. andLesk, A. M., J.Mol.Biol., 196,901 (1987); Chothia, C. et al., Nature.December 21-28; 342(6252):877-83 (1989); Kabat E. A. et al., NationalInstitutes of Health, Bethesda, Md. (1991)). The three CDRs areinterposed between flanking stretches known as framework regions (FRs),which are more highly conserved than the CDRs and form a scaffold tosupport the hypervariable loops. The constant regions of the heavy andlight chains are not involved in antigen binding, but exhibit variouseffector functions. Antibodies are assigned to classes based on theamino acid sequence of the constant region of their heavy chain. Thefive major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, andIgM, which are characterized by the presence of α, δ, ε, γ, and μ heavychains, respectively. Several of the major antibody classes are dividedinto subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain),IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), orIgA2 (α2 heavy chain).

The term “antigen-binding fragment” as used herein refers to an antibodyfragment formed from a portion of an antibody comprising one or moreCDRs, or any other antibody fragment that binds to an antigen but doesnot comprise an intact native antibody structure. Examples ofantigen-binding fragment include, without limitation, a diabody, a Fab,a Fab′, a F(ab′)₂, an Fv fragment, a disulfide stabilized Fv fragment(dsFv), a (dsFv)₂, a bispecific dsFv (dsFv-dsFv′), a disulfidestabilized diabody (ds diabody), a single-chain antibody molecule(scFv), an scFv dimer (bivalent diabody), a multispecific antibody, acamelized single domain antibody, a nanobody, a domain antibody, and abivalent domain antibody. An antigen-binding fragment is capable ofbinding to the same antigen to which the parent antibody binds. Incertain embodiments, an antigen-binding fragment may comprise one ormore CDRs from a particular human antibody grafted to a framework regionfrom one or more different human antibodies.

“Fab” with regard to an antibody refers to that portion of the antibodyconsisting of a single light chain (both variable and constant regions)bound to the variable region and first constant region of a single heavychain by a disulfide bond.

“Fab′” refers to a Fab fragment that includes a portion of the hingeregion.

“F(ab′)₂” refers to a dimer of Fab′.

“Fc” with regard to an antibody refers to that portion of the antibodyconsisting of the second and third constant regions of a first heavychain bound to the second and third constant regions of a second heavychain via disulfide bonding. The Fc portion of the antibody isresponsible for various effector functions such as ADCC, and CDC, butdoes not function in antigen binding.

“Fv” with regard to an antibody refers to the smallest fragment of theantibody to bear the complete antigen binding site. An Fv fragmentconsists of the variable region of a single light chain bound to thevariable region of a single heavy chain.

“Single-chain Fv antibody” or “scFv” refers to an engineered antibodyconsisting of a light chain variable region and a heavy chain variableregion connected to one another directly or via a peptide linkersequence (Huston J S et al. Proc Natl Acad Sci USA, 85:5879(1988)).

“Single-chain Fv-Fc antibody” or “scFv-Fc” refers to an engineeredantibody consisting of a scFv connected to the Fc region of an antibody.

“Camelized single domain antibody,” “heavy chain antibody,” or “HCAb”refers to an antibody that contains two V_(H) domains and no lightchains (Riechmann L. and Muyldermans S., J Immunol Methods. December 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. June; 74(4):277-302(2001); WO94/04678; WO94/25591; U.S. Pat. No. 6,005,079). Heavy chainantibodies were originally derived from Camelidae (camels, dromedaries,and llamas). Although devoid of light chains, camelized antibodies havean authentic antigen-binding repertoire (Hamers-Casterman C. et al.,Nature. June 3; 363(6428):446-8 (1993); Nguyen V K. et al. “Heavy-chainantibodies in Camelidae; a case of evolutionary innovation,”Immunogenetics. April; 54(1):39-47 (2002); Nguyen V K. et al.Immunology. May; 109(1):93-101 (2003)). The variable domain of a heavychain antibody (VHH domain) represents the smallest knownantigen-binding unit generated by adaptive immune responses (Koch-NolteF. et al., FASEB J. November; 21(13):3490-8. Epub 2007 Jun. 15 (2007)).

A “nanobody” refers to an antibody fragment that consists of a VHHdomain from a heavy chain antibody and two constant domains, CH2 andCH3.

“Diabodies” include small antibody fragments with two antigen-bindingsites, wherein the fragments comprise a V_(H) domain connected to aV_(L) domain in the same polypeptide chain (V_(H)-V_(L) or V_(L)-V_(H))(see, e.g., Holliger P. et al., Proc Natl Acad Sci USA. July 15;90(14):6444-8 (1993); EP404097; WO93/11161). By using a linker that istoo short to allow pairing between the two domains on the same chain,the domains are forced to pair with the complementary domains of anotherchain, thereby creating two antigen-binding sites. The antigen-bindingsites may target the same of different antigens (or epitopes).

A “domain antibody” refers to an antibody fragment containing only thevariable region of a heavy chain or the variable region of a lightchain. In certain instances, two or more V_(H) domains are covalentlyjoined with a peptide linker to create a bivalent or multivalent domainantibody. The two V_(H) domains of a bivalent domain antibody may targetthe same or different antigens.

In certain embodiments, a “(dsFv)₂” comprises three peptide chains: twoV_(H) moieties linked by a peptide linker and bound by disulfide bridgesto two V_(L) moieties.

In certain embodiments, a “bispecific ds diabody” comprisesV_(H1)-V_(L2) (linked by a peptide linker) bound to V_(L1)-V_(H2) (alsolinked by a peptide linker) via a disulfide bridge between V_(H1) andV_(L1).

In certain embodiments, a “bispecific dsFv” or dsFv-dsFv′” comprisesthree peptide chains: a V_(H1)-V_(H2) moiety wherein the heavy chainsare linked by a peptide linker (e.g., a long flexible linker) and boundto V_(L1) and V_(L2) moieties, respectively, via disulfide bridges,wherein each disulfide paired heavy and light chain has a differentantigen specificity.

In certain embodiments, an “scFv dimer” is a bivalent diabody orbivalent ScFv (B sFv) comprising V_(H)-V_(L) (linked by a peptidelinker) dimerized with another V_(H)-V_(L) moiety such that V_(H)'s ofone moiety coordinate with the V_(L)'s of the other moiety and form twobinding sites which can target the same antigens (or eptipoes) ordifferent antigens (or eptipoes). In other embodiments, an “scFv dimer”is a bispecific diabody comprising V_(H1)-V_(L2) (linked by a peptidelinker) associated with V_(L1)-V_(H2) (also linked by a peptide linker)such that V_(H1) and V_(L1) coordinate and V_(H2) and V_(L2) coordinateand each coordinated pair has a different antigen specificity.

The term “fully human” as used herein, with reference to antibody orantigen-binding fragment, means that the antibody or the antigen-bindingfragment has or consists of amino acid sequence(s) corresponding to thatof an antibody produced by a human or a human immune cell, or derivedfrom a non-human source such as a transgenic non-human animal thatutilizes human antibody repertoires or other human antibody-encodingsequences. In certain embodiments, a fully human antibody does notcomprise amino acid residues (in particular antigen-binding residues)derived from a non-human antibody.

The term “humanized” as used herein, with reference to antibody orantigen-binding fragment, means that the antibody or the antigen-bindingfragment comprises CDRs derived from non-human animals, FR regionsderived from human, and when applicable, the constant regions derivedfrom human. A humanized antibody or antigen-binding fragment is usefulas human therapeutics in certain embodiments because it has reducedimmunogenicity in human. In some embodiments, the non-human animal is amammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guineapig, or a hamster. In some embodiments, the humanized antibody orantigen-binding fragment is composed of substantially all humansequences except for the CDR sequences which are non-human. In someembodiments, the FR regions derived from human may comprise the sameamino acid sequence as the human antibody from which it is derived, orit may comprise some amino acid changes, for example, no more than 10,9, 8, 7, 6, 5, 4, 3, 2, or 1 changes of amino acid. In some embodiments,such change in amino acid could be present in heavy chain FR regionsonly, in light chain FR regions only, or in both chains. In somepreferable embodiments, the humanized antibodies comprise human FR1-3and human JH and Jκ.

The term “chimeric” as used herein, means an antibody or antigen-bindingfragment, having a portion of heavy and/or light chain derived from onespecies, and the rest of the heavy and/or light chain derived from adifferent species. In an illustrative example, a chimeric antibody maycomprise a constant region derived from human and a variable region froma non-human species, such as from mouse.

“PD-1” as used herein refers programmed cell death protein, whichbelongs to the superfamily of immunoglobulin and functions ascoinhibitory receptor to negatively regulate the immune system. PD-1 isa member of the CD28/CTLA-4 family, and has two known ligands includingPD-L1 and PD-L2. Representative amino acid sequence of human PD-1 isdisclosed under the NCBI accession number: NP_005009.2, and therepresentative nucleic acid sequence encoding the human PD-1 is shownunder the NCBI accession number: NM_005018.2.

“PD-L1” as used herein refers to programmed cell death ligand 1 (PD-L1,see, for example, Freeman et al. (2000) J. Exp. Med. 192:1027).Representative amino acid sequence of human PD-L1 is disclosed under theNCBI accession number: NP_054862.1, and the representative nucleic acidsequence encoding the human PD-L1 is shown under the NCBI accessionnumber: NM_014143.3. PD-L1 is expressed in placenta, spleen, lymphnodes, thymus, heart, fetal liver, and is also found on many tumor orcancer cells. PD-L1 binds to its receptor PD-1 or B7-1, which isexpressed on activated T cells, B cells and myeloid cells. The bindingof PD-L1 and its receptor induces signal transduction to suppressTCR-mediated activation of cytokine production and T cell proliferation.Accordingly, PD-L1 plays a major role in suppressing immune systemduring particular events such as pregnancy, autoimmune diseases, tissueallografts, and is believed to allow tumor or cancer cells to circumventthe immunological checkpoint and evade the immune response.

“Anti-PD-1 antibody” as used herein refers to an antibody that iscapable of specific binding to PD-1 (e.g. human or monkey PD-1) with anaffinity which is sufficient to provide for diagnostic and/ortherapeutic use.

The term “specific binding” or “specifically binds” as used hereinrefers to a non-random binding reaction between two molecules, such asfor example between an antibody and an antigen. In certain embodiments,the antibodies or antigen-binding fragments provided herein specificallybind human and/or monkey PD-1 with a binding affinity (K_(D)) of ≤10⁻⁶ M(e.g., ≤5×10⁻⁷ M, ≤2×10⁻⁷M, ≤10⁻⁷M, ≤5×10⁻⁸ M, ≤2×10⁻⁸ M, ≤10⁻⁸ M,≤5×10⁻⁹ M, ≤2×10⁻⁹ M, ≤10⁻⁹M, 10⁻¹⁰ M). K_(D) as used herein refers tothe ratio of the dissociation rate to the association rate(k_(off)/k_(on)), may be determined using surface plasmon resonancemethods for example using instrument such as Biacore.

The ability to “block binding” or “compete for the same epitope” as usedherein refers to the ability of an antibody or antigen-binding fragmentto inhibit the binding interaction between two molecules (e.g. humanPD-1 and an anti-PD-1 antibody) to any detectable degree. In certainembodiments, an antibody or antigen-binding fragment that blocks bindingbetween two molecules inhibits the binding interaction between the twomolecules by at least 50%. In certain embodiments, this inhibition maybe greater than 60%, greater than 70%, greater than 80%, or greater than90%.

The term “epitope” as used herein refers to the specific group of atomsor amino acids on an antigen to which an antibody binds. Two antibodiesmay bind the same epitope within an antigen if they exhibit competitivebinding for the antigen. For example, if an antibody or antigen-bindingfragment as disclosed herein blocks binding of the exemplary antibodiessuch as 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.103.11-v2 hAb, 1.139.15hAb, and 1.153.7 hAb to human PD-1, then the antibody or antigen-bindingfragment may be considered to bind the same epitope as those exemplaryantibodies.

A particular amino acid residue within the epitope can be mutated, e.g.by alanine scanning mutagenesis, and mutations that reduce or preventprotein binding are identified. An “alanine scanning mutagenesis” is amethod that can be performed for identifying certain residues or regionsof a protein that affect the interaction of the epitope with anothercompound or protein that binds to it. A residue or group of targetresidues within the protein is replaced by a neutral or negativelycharged amino acid (most preferably alanine or polyalanine, or aconservative amino acid substitution). Any mutation of the amino acidresidues or codons encoding the same that reduces binding of the proteinmore than a threshold or reduces binding of the protein to the maximaldegree than other mutations is likely to be within the epitope bound bythe protein. In certain embodiments of the present disclosure, theepitope that is critical for the PD-1 antibody comprises at least one ofthe amino acid residues of V64, P83, D85, L128, A129, P130, K131, A132and Q133.

“1.7.3 hAb” as used herein refers to a fully human monoclonal antibodyhaving a heavy chain variable region of SEQ ID NO: 45, light chainvariable region of SEQ ID NO: 47, and a human constant region of IgG4isotype.

“1.49.9 hAb” as used herein refers to a fully human monoclonal antibodyhaving a heavy chain variable region of SEQ ID NO: 49, light chainvariable region of SEQ ID NO: 51, and a human constant region of IgG4isotype.

“1.103.11 hAb” as used herein refers to a fully human monoclonalantibody having a heavy chain variable region of SEQ ID NO: 53, lightchain variable region of SEQ ID NO: 55, and a human constant region ofIgG4 isotype.

“1.103.11-v2 hAb” as used herein refers to a fully human monoclonalantibody having a heavy chain variable region of SEQ ID NO: 53, lightchain variable region of SEQ ID NO: 67, and a human constant region ofIgG4 isotype.

“1.139.15 hAb” as used herein refers to a fully human monoclonalantibody having a heavy chain variable region of SEQ ID NO: 57, lightchain variable region of SEQ ID NO: 59, and a human constant region ofIgG4 isotype.

“1.153.7 hAb” as used herein refers to a fully human monoclonal antibodyhaving a heavy chain variable region of SEQ ID NO: 61, light chainvariable region of SEQ ID NO: 63, and a human constant region of IgG4isotype.

A “conservative substitution” with reference to amino acid sequencerefers to replacing an amino acid residue with a different amino acidresidue having a side chain with similar physiochemical properties. Forexample, conservative substitutions can be made among amino acidresidues with hydrophobic side chains (e.g. Met, Ala, Val, Leu, andIle), among residues with neutral hydrophilic side chains (e.g. Cys,Ser, Thr, Asn and Gln), among residues with acidic side chains (e.g.Asp, Glu), among amino acids with basic side chains (e.g. His, Lys, andArg), or among residues with aromatic side chains (e.g. Trp, Tyr, andPhe). As known in the art, conservative substitution usually does notcause significant change in the protein conformational structure, andtherefore could retain the biological activity of a protein.

“Percent (%) sequence identity” with respect to amino acid sequence (ornucleic acid sequence) is defined as the percentage of amino acid (ornucleic acid) residues in a candidate sequence that are identical to theamino acid (or nucleic acid) residues in a reference sequence, afteraligning the sequences and, if necessary, introducing gaps, to achievethe maximum number of identical amino acids (or nucleic acids).Conservative substitution of the amino acid residues may or may not beconsidered as identical residues. Alignment for purposes of determiningpercent amino acid (or nucleic acid) sequence identity can be achieved,for example, using publicly available tools such as BLASTN, BLASTp(available on the website of U.S. National Center for BiotechnologyInformation (NCBI), see also, Altschul S. F. et al, J. Mol. Biol.,215:403-410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402(1997)), ClustalW2 (available on the website of European BioinformaticsInstitute, see also, Higgins D. G. et al, Methods in Enzymology,266:383-402 (1996); Larkin M. A. et al, Bioinformatics (Oxford,England), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR)software. Those skilled in the art may use the default parametersprovided by the tool, or may customize the parameters as appropriate forthe alignment, such as for example, by selecting a suitable algorithm.

“T cell” as used herein includes CD4⁺ T cells, CD8⁺ T cells, T helper 1type T cells, T helper 2 type T cells, T helper 17 type T cells andinhibitory T cells.

“Effector functions” as used herein refer to biological activitiesattributable to the binding of Fc region of an antibody to its effectorssuch as C1 complex and Fc receptor. Exemplary effector functionsinclude: complement dependent cytotoxicity (CDC) induced by interactionof antibodies and C1q on the C1 complex; antibody-dependentcell-mediated cytotoxicity (ADCC) induced by binding of Fc region of anantibody to Fc receptor on an effector cell; and phagocytosis.

“Cancer” or “cancerous condition” as used herein refers to any medicalcondition mediated by neoplastic or malignant cell growth,proliferation, or metastasis, and includes both solid cancers andnon-solid cancers such as leukemia. “Tumor” as used herein refers to asolid mass of neoplastic and/or malignant cells.

“Treating” or “treatment” of a condition as used herein includespreventing or alleviating a condition, slowing the onset or rate ofdevelopment of a condition, reducing the risk of developing a condition,preventing or delaying the development of symptoms associated with acondition, reducing or ending symptoms associated with a condition,generating a complete or partial regression of a condition, curing acondition, or some combination thereof. With regard to cancer,“treating” or “treatment” may refer to inhibiting or slowing neoplasticor malignant cell growth, proliferation, or metastasis, preventing ordelaying the development of neoplastic or malignant cell growth,proliferation, or metastasis, or some combination thereof. With regardto a tumor, “treating” or “treatment” includes eradicating all or partof a tumor, inhibiting or slowing tumor growth and metastasis,preventing or delaying the development of a tumor, or some combinationthereof.

An “isolated” substance has been altered by the hand of man from thenatural state. If an “isolated” composition or substance occurs innature, it has been changed or removed from its original environment, orboth. For example, a polynucleotide or a polypeptide naturally presentin a living animal is not “isolated,” but the same polynucleotide orpolypeptide is “isolated” if it has been sufficiently separated from thecoexisting materials of its natural state so as to exist in asubstantially pure state. In certain embodiments, the antibodies andantigen-binding fragments have a purity of at least 90%, 93%, 95%, 96%,97%, 98%, 99% as determined by electrophoretic methods (such asSDS-PAGE, isoelectric focusing, capillary electrophoresis), orchromatographic methods (such as ion exchange chromatography or reversephase HPLC).

The term “vector” as used herein refers to a vehicle into which apolynucleotide encoding a protein may be operably inserted so as tobring about the expression of that protein. A vector may be used totransform, transduce, or transfect a host cell so as to bring aboutexpression of the genetic element it carries within the host cell.Examples of vectors include plasmids, phagemids, cosmids, artificialchromosomes such as yeast artificial chromosome (YAC), bacterialartificial chromosome (BAC), or P1-derived artificial chromosome (PAC),bacteriophages such as lambda phage or M13 phage, and animal viruses.Categories of animal viruses used as vectors include retrovirus(including lentivirus), adenovirus, adeno-associated virus, herpesvirus(e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, andpapovavirus (e.g., SV40). A vector may contain a variety of elements forcontrolling expression, including promoter sequences, transcriptioninitiation sequences, enhancer sequences, selectable elements, andreporter genes. In addition, the vector may contain an origin ofreplication. A vector may also include materials to aid in its entryinto the cell, including but not limited to a viral particle, aliposome, or a protein coating.

The phrase “host cell” as used herein refers to a cell into which anexogenous polynucleotide and/or a vector has been introduced.

A “disease associated with or related to PD-1” as used herein refers toany condition that is caused by, exacerbated by, or otherwise linked toincreased or decreased expression or activities of PD-1 (e.g. a humanPD-1).

The term “therapeutically effective amount” or “effective dosage” asused herein refers to the dosage or concentration of a drug effective totreat a disease or condition associated with human PD-1. For example,with regard to the use of the antibodies or antigen-binding fragmentsdisclosed herein to treat cancer, a therapeutically effective amount isthe dosage or concentration of the antibody or antigen-binding fragmentcapable of eradicating all or part of a tumor, inhibiting or slowingtumor growth, inhibiting growth or proliferation of cells mediating acancerous condition, inhibiting tumor cell metastasis, ameliorating anysymptom or marker associated with a tumor or cancerous condition,preventing or delaying the development of a tumor or cancerouscondition, or some combination thereof.

The term “pharmaceutically acceptable” indicates that the designatedcarrier, vehicle, diluent, excipient(s), and/or salt is generallychemically and/or physically compatible with the other ingredientscomprising the formulation, and physiologically compatible with therecipient thereof.

Anti-PD-1 Antibody

In one aspect, the present disclosure provides anti-PD-1 antibodies andthe antigen-binding fragments thereof. PD-1, also called as CD279, isknown as a key immune-checkpoint receptor expressed by activated Tcells, which mediates immunosuppression. PD-1 ligand 1 (PD-L1) is a 40kDa transmembrane protein expressed on various tumor cells, stromalcells or both, and binds to PD-1. Inhibition of the interaction betweenPD-1 and PD-L1 can enhance T-cell responses and thus mediatesanti-cancer activity.

In certain embodiments, the present disclosure provides exemplary fullyhuman monoclonal antibodies 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb,1.103.11-v2 hAb, 1.139.15 hAb, and 1.153.7 hAb, whose CDR sequences areshown in the below Table 1, and heavy or light chain variable regionsequences are also shown below.

TABLE 1 CDR1 CDR2 CDR3 1.7.3 hAb- SEQ ID NO: 1 SEQ ID NO: 3 SEQ ID NO: 5VH(23466-VH) STTYYWV SISYSGNTYYNPSLKS HLGYNGRYLPFDY SEQ ID NO: 2SEQ ID NO: 4 SEQ ID NO: 6 AGT ACT ACT TAC AGT ATC TCT TAT AGTCAT CTA GGG TAT TAC TGG GTC GGG AAC ACC TAC TAC AAT GGG AGG TACAAT CCG TCC CTC AAG CTC CCC TTT GAC AGT TAC 1.7.3 hAb- SEQ ID NO: 7SEQ ID NO: 9 SEQ ID NO: 11 VL(23195-VL) TGTSSDVGFYNYVS DVTNRPSSSYTSISTWV SEQ ID NO: 8 SEQ ID NO: 10 SEQ ID NO: 12 ACT GGA ACC AGCGAT GTC ACT AAT CGG AGC TCA TAT ACA AGT GAC GTT GGT CCC TCAAGC ATC AGC ACT TTT TAT AAC TAT TGG GTG GTC TCC 1.49.9 hAb SEQ ID NO: 13SEQ ID NO: 15 SEQ ID NO: 5 VH(20951-VH) SSTYYWG SISYSGSTYYNPSLKSHLGYNGRYLPFDY SEQ ID NO: 14 SEQ ID NO: 16 SEQ ID NO: 6 AGT AGT ACT TACAGT ATC TCT TAT AGT CAT CTA GGG TAT TAC TGG GGC GGG AGC ACC TAC TACAAT GGG AGG TAC AAT CCG TCC CTC AAG CTC CCC TTT GAC AGT TAC 1.49.9 hAb-SEQ ID NO: 7 SEQ ID NO: 17 SEQ ID NO 11 VH(20951-VL) TGTSSDVGFYNYVSDVSNRPS SSYTSISTWV SEQ ID NO: 8 SEQ ID NO: 18 SEQ ID NO: 12ACT GGA ACC AGC CGG CCC TCA GAT GTC AGC TCA TAT ACA AGT GAC GTT GGTAGT AAT AGC ATC AGC ACT TTT TAT AAC TAT TGG GTG GTC TCC 1.103.11 hAb-SEQ ID NO: 1 SEQ ID NO: 15 SEQ ID NO: 5 VH(20975-VH) STTYYWVSISYSGSTYYNPSLKS HLGYNGRYLPFDY SEQ ID NO: 2 SEQ ID NO: 16 SEQ ID NO: 6AGT ACT ACT TAC AGT ATC TCT TAT AGT CAT CTA GGG TAT TAC TGG GTCGGG AGC ACC TAC TAC AAT GGG AGG TAC AAT CCG TCC CTC AAG CTC CCC TTT GACAGT TAC 1.103.11 hAb- SEQ ID NO: 7 SEQ ID NO: 17 SEQ ID NO: 19VH(20975-VL) TGTSSDVGFYNYVS DVSNRPS SSYTNISTWV SEQ ID NO: 8SEQ ID NO: 18 SEQ ID NO: 20 ACT GGA ACC AGC GAT GTC AGT AAT CGGAGC TCA TAT ACA AGT GAC GTT GGT CCC TCA AAC ATC AGC ACT TTT TAT AAC TATTGG GTG GTC TCC 1.139.15 hAb- SEQ ID NO: 21 SEQ ID NO: 23 SEQ ID NO: 25VH(23521-VH) STTYYWG SISYSGTTYYNPSLKS HLGYNSNWYPFDY SEQ ID NO: 22SEQ ID NO: 24 SEQ ID NO: 26 AGT ACT ACT TAC AGT ATC TCT TAT AGTCAT CTC GGG TAT TAC TGG GGC GGG ACC ACC TAC TAC AAC AGC AAC TGGAAC CCG TCC CTC AAG TAC CCT TTT GAC AGT TAC 1.139.15 hAb- SEQ ID NO: 27SEQ ID NO: 29 SEQ ID NO: 31 VH(23521-VL) TGTSSDVGSYNRVS EVSNRPSSSYTSSSTWV SEQ ID NO: 28 SEQ ID NO: 30 SEQ ID NO: 32 ACT GGA ACC AGCGAG GTC AGT AAT CGG AGC TCA TAT ACA AGT GAC GTT GGT CCC TCAAGC AGC AGC ACT AGT TAT AAC CGT TGG GTG GTC TCC 1.153.7 hAb-SEQ ID NO: 33 SEQ ID NO: 35 SEQ ID NO: 37 VH(20942-VH) SHAMSTITGGGGSIYYADSVKG NRAGEGYFDY SEQ ID NO: 34 SEQ ID NO: 36 SEQ ID NO: 38AGC CAT GCC ATG ACT ATT ACT GGT GGT AAC CGC GCT GGG AGC GGT GGT AGC ATAGAG GGT TAC TTT TAC TAC GCA GAC TCC GAC TAC GTG AAG GGC 1.153.7 hAb-SEQ ID NO: 39 SEQ ID NO: 41 SEQ ID NO: 43 VH(20942-VL) GGDNIGNKDVHRDSNRPS QVWDSIWV SEQ ID NO: 40 SEQ ID NO: 42 SEQ ID NO: 44GGG GGA GAC AAC AGG GAT AGC AAC CGG CAG GTG TGG GAC ATT GGA AAT AAACCC TCT AGC ATT TGG GTG GAT GTG CAC 1.103.11-v2 SEQ ID NO: 1SEQ ID NO: 15 SEQ ID NO: 5 hAb- STTYYWV SISYSGSTYYNPSLKS HLGYNGRYLPFDYVH(20975-VH) SEQ ID NO: 2 SEQ ID NO: 16 SEQ ID NO: 6 AGT ACT ACT TACAGT ATC TCT TAT AGT CAT CTA GGG TAT TAC TGG GTC GGG AGC ACC TACAAT GGG AGG TAC TAC AAT CCG TCC CTC CTC CCC TTT GAC AAG AGT TAC1.103.11- SEQ ID NO: 7 SEQ ID NO: 17 SEQ ID NO: 65 v2 hAb-TGTSSDVGFYNYVS DVSNRPS SSYTSISTWV VH(20975- SEQ ID NO: 8 SEQ ID NO: 18SEQ ID NO: 66 2-VL) ACT GGA ACC AGC GAT GTC AGT AAT CGG AGC TCA TAT ACAAGT GAC GTT GGT CCC TCA AGC ATC AGC ACT TTT TAT AAC TAT TGG GTG GTC TCC

1.7.3 hAb-VH(23466-VH): (SEQ ID NO:45 for amino acid and SEQ ID NO:46for nucleic acid) with heavy chain CDRs1-3: SEQ ID NOs: 1, 3, 5 areamino acid sequences and SEQ ID NO:2, 4, 6 are nucleic acid sequences,respectively:

V segment: IGHV4-39*01 D segment: IGHD1-26*01 J segment: IGHJ4*02 Q   L   Q   L   Q   E   S   G   P   G   L   V   K   P   S   1CAG CTG CAG CTG CAG GAG TCG GGC CCA GGA CTG GTG AAG CCT TCG E   T   L   T   L   T   C   T   V   S   G   D   S   I   S  46GAG ACC CTG ACC CTC ACC TGC ACT GTC TCT GGT GAC TCC ATC AGC           CDR1  -------------------------- S   T   T   Y   Y   W   V   W   I   R   Q   P   P   G   K  91AGT ACT ACT TAC TAC TGG GTC TGG ATC CGC CAG CCC CCA GGG AAG                                          CDR2                         --------------------------------- G   L   E   W   I   G   S   I   S   Y   S   G   N   T   Y 136GGA CTG GAG TGG ATT GGG AGT ATC TCT TAT AGT GGG AAC ACC TAC             CDR2  ------------------------- Y   N   P   S   L   K   S   R   V   T   I   S   V   D   T 181TAC AAT CCG TCC CTC AAG AGT CGA GTC ACC ATA TCC GTA GAC ACG S   K   N   H   F   S   L   K   L   S   S   V   A   A   T 226TCC AAG AAC CAC TTC TCC CTG AAG CTG AGT TCT GTG GCC GCC ACA                                              CDR3                                     --------------------- D   T   A   L   Y   Y   C   A   R   H   L   G   Y   N   G 271GAC ACG GCT CTA TAT TAC TGT GCG AGA CAT CTA GGG TAT AAT GGG             CDR3  ------------------------- R   Y   L   P   F   D   Y   W   G   Q   G   T   L   V   T 316AGG TAC CTC CCC TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC V   S   S  (SEQ ID NO: 45) 361 GTC TCC TCC (SEQ ID NO: 46)

1.7.3 hAb-VL(23195-VL): (SEQ ID NO:47 for amino acid and SEQ ID NO:48for nucleic acid) with light chain CDRs1-3: SEQ ID NOs: 7, 9, 11 areamino acid sequences and SEQ ID NO:8, 10, 12 are nucleic acid sequences,respectively:

V segment: IGLV2-14*01 J segment: IGLJ3*02 Q   S   A   L   T   Q   P   A   S   V   S   G   S   P   G   1CAG TCT GCC CTG ACT CAG CCT GCC TCC GTG TCT GGG TCT CCT GGA                                         CDR1                             ---------------------------- Q   S   I   T   I   S   C   T   G   T   S   S   D   V   G  46CAG TCG ATC ACC ATC TCC TGC ACT GGA ACC AGC AGT GAC GTT GGT         CDR1 --------------------- F   Y   N   Y   V   S   W   Y   Q   Q   H   P   G   K   A  91  TTT TAT AAC TAT GTC TCC TGG TAC CAA CAG CAC CCA GGC AAA GCC                                    CDR2                         ------------------------- P   E   L   M   I   Y   D   V   T   N   R   P   S   G   V 136CCC GAA CTC ATG ATT TAT GAT GTC ACT AAT CGG CCC TCA GGG GTT S   D   R   F   S   G   S   K   S   G   N   T   A   S   L 181TCT GAT CGC TTC TCT GGC TCC AAG TCT GGC AAC ACG GCC TCC CTG T   I   S   G   L   Q   A   E   D   E   A   D   Y   Y   C 226ACC ATC TCT GGG CTC CAG GCT GAG GAC GAG GCT GAT TAT TAC TGC                 CDR3  ------------------------------------- S   S   Y   T   S   I   S   T   W   V   F   G   G   G   T 261AGC TCA TAT ACA AGC ATC AGC ACT TGG GTG TTC GGC GGA GGG ACC K   L   T   V   L  (SEQ ID NO: 47) 326AAG CTG ACC GTC CTA (SEQ ID NO: 48)

1.49.9 hAb-VH(20951-VH): (SEQ ID NO:49 for amino acid and SEQ ID NO:50for nucleic acid) with heavy chain CDRs 1-3: SEQ ID NOs: 13, 15, 5 areamino acid sequences and SEQ ID NO:14, 16, 6 are nucleic acid sequences,respectively:

V segment: IGHV4-39*01 D segment: IGHD1-26*01 J segment: IGHJ4*02 Q   L   Q   L   Q   E   S   G   P   G   L   V   K   P   S   1CAG CTG CAG CTG CAG GAG TCG GGC CCA GGA CTG GTG AAG CCT TCG E   T   L   S   L   T   C   T   V   S   G   G   S   I   S  46GAG ACC CTG TCC CTC ACC TGC ACT GTC TCT GGT GGC TCC ATC AGC           CDR1  ------------------------- S   S   T   Y   Y   W   G   W   I   R   Q   P   P   G   K  91AGT AGT ACT TAC TAC TGG GGC TGG ATC CGC CAG CCC CCA GGG AAG                                       CDR2                         --------------------------------- G   L   E   W   I   G   S   I   S   Y   S   G   S   T   Y 136GGA CTG GAG TGG ATT GGG AGT ATC TCT TAT AGT GGG AGC ACC TAC           CDR2  ------------------------- Y   N   P   S   L   K   S   R   V   T   I   S   V   D   T 181TAC AAT CCG TCC CTC AAG AGT CGA GTC ACC ATA TCC GTA GAC ACG S   K   N   Q   F   S   L   K   L   S   S   V   T   D   A 226TCC AAG AAC CAG TTC TCC CTG AAG CTG AGC TCT GTG ACC GAC GCA                                               CDR3                                     ---------------------  D   T   A   V   Y   Y   C   A   R   H   L   G   Y   N   G 261GAC ACG GCT GTG TAT TAC TGT GCG AGA CAT CTA GGG TAT AAT GGG           CDR3  ------------------------- R   Y   L   P   F   D   Y   W   G   Q   G   T   L   V   T 316AGG TAC CTC CCC TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC V   S   S  (SEQ ID NO: 49) 361 GTC TCC TCC (SEQ ID NO: 50)

1.49.9 hAb-VL(21526-VL): (SEQ ID NO:51 for amino acid and SEQ ID NO:52for nucleic acid) with light chain CDRs 1-3: SEQ ID NOs: 7, 17, 11 areamino acid sequences and SEQ ID NO:8, 18, 12 are nucleic acid sequences,respectively:

V segment: IGLV2-14*01 J segment: IGLJ3*02 Q   S   A   L   T   Q   P   A   S   V   S   G   S   P   G   1CAG TCT GCC CTG ACT CAG CCT GCC TCC GTG TCT GGG TCT CCT GGA                                         CDR1                             ----------------------------- Q   S   I   T   I   S   C   T   G   T   S   S   D   V   G  46CAG TCG ATC ACC ATC TCC TGC ACT GGA ACC AGC AGT GAC GTT GGT         CDR1  --------------------- F   Y   N   Y   V   S   W   Y   Q   Q   H   P   G   K   A  91TTT TAT AAC TAT GTC TCC TGG TAC CAA CAG CAC CCA GGC AAA GCC                                   CDR2                         ------------------------- P   E   V   M   I   Y   D   V   S   N   R   P   S   G   V 136CCC GAA GTC ATG ATT TAT GAT GTC AGT AAT CGG CCC TCA GGG GTT S   D   R   F   S   G   S   K   S   G   N   T   A   S   L 181TCT GAT CGC TTC TCT GGC TCC AAG TCT GGC AAC ACG GCC TCC CTG T   I   S   G   L   Q   A   E   D   E   A   D   Y   Y   C 226ACT ATC TCT GGG CTC CAG GCT GAG GAC GAG GCT GAT TAT TAC TGC               CDR3  ------------------------------------- S   S   Y   T   S   I   S   T   W   V   F   G   G   G   T 261AGC TCA TAT ACA AGC ATC AGC ACT TGG GTG TTC GGC GGA GGG ACC K   L   T   V   L  (SEQ ID NO: 51) 316AAG CTG ACT GTC CTA (SEQ ID NO: 52)

1.103.11 hAb-VH(20975-VH): (SEQ ID NO:53 for amino acid and SEQ ID NO:54for nucleic acid) with heavy chain CDRs 1-3: SEQ ID NOs: 1, 15, 5 areamino acid sequences and SEQ ID NO:2, 16, 6 are nucleic acid sequences,respectively:

V segment: IGHV4-39*01 D segment: IGHD1-26*01 J segment: IGHJ4*02 Q   L   Q   L   Q   E   S   G   P   G   L   V   K   P   S   1CAG CTG CAG CTG CAG GAG TCG GGC CCA GGA CTG GTG AAG CCT TCG E   T   L   T   L   T   C   T   V   S   A   D   S   I   S  46GAG ACC CTG ACC CTC ACC TGC ACT GTC TCT GCT GAC TCC ATC AGC            CDR1  ------------------------- S   T   T   Y   Y   W   V   W   I   R   Q   P   P   G   K  91AGT ACT ACT TAC TAC TGG GTC TGG ATC CGC CAG CCC CCA GGG AAG                                        CDR2                         --------------------------------- G   L   E   W   I   G   S   I   S   Y   S   G   S   T   Y 136GGA CTG GAG TGG ATT GGG AGT ATC TCT TAT AGT GGG AGC ACC TAC            CDR2  ------------------------- Y   N   P   S   L   K   S   R   V   T   V   S   V   D   T 181TAC AAT CCG TCC CTC AAG AGT CGA GTC ACC GTA TCC GTA GAC ACG S   K   N   Q   F   S   L   K   L   N   S   V   A   A   T 226TCC AAG AAC CAG TTC TCC CTG AAG CTG AAC TCT GTG GCC GCC ACA                                            CDR3                                     --------------------- D   T   A   L   Y   Y   C   A   R   H   L   G   Y   N   G 261GAC ACG GCT CTA TAT TAC TGT GCG AGA CAT CTA GGG TAT AAT GGG           CDR3  ------------------------- R   Y   L   P   F   D   Y   W   G   Q   G   T   L   V   T 316AGG TAC CTC CCC TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC V   S   S  (SEQ ID NO: 53) 361 GTC TCC TCC (SEQ ID NO: 54)

1.103.11 hAb-VL(21038-VL): (SEQ ID NO:55 for amino acid and SEQ ID NO:56for nucleic acid) with light chain CDRs 1-3: SEQ ID NOs: 7, 17, 19 areamino acid sequences and SEQ ID NO:8, 18, 20 are nucleic acid sequences,respectively:

V segment: IGLV2-14*01 J segment: IGLJ3*02 Q   S   A   L   T   Q   P   A   S   V   S   G   S   P   G   1CAG TCT GCC CTG ACT CAG CCT GCC TCC GTG TCT GGG TCT CCT GGA                                        CDR1                             ----------------------------- Q   S   I   T   I   S   C   T   G   T   S   S   D   V   G  46CAG TCG ATC ACC ATC TCC TGC ACT GGA ACC AGC AGT GAC GTT GGT         CDR1 --------------------- F   Y   N   Y   V   S   W   Y   Q   Q   H   P   G   K   A  91TTT TAT AAC TAT GTC TCC TGG TAC CAA CAG CAC CCA GGC AAA GCC                                    CDR2                         ------------------------- P   E   L   M   I   Y   D   V   S   N   R   P   S   G   V 136CCC GAA CTC ATG ATT TAT GAT GTC AGT AAT CGG CCC TCA GGG GTT S   D   R   F   S   G   S   K   S   G   N   T   A   S   L 181TCT GAT CGC TTC TCT GGC TCC AAG TCT GGC AAC ACG GCC TCC CTG T   I   S   G   L   Q   A   E   D   E   A   D   Y   Y   C 226ACC ATC TCT GGG CTC CAG GCT GAG GAC GAG GCT GAT TAT TAC TGC                CDR3  ------------------------------------- S   S   Y   T   N   I   S   T   W   V   F   G   G   G   T 261AGC TCA TAT ACA AAC ATC AGC ACT TGG GTG TTC GGC GGA GGG ACC K   L   T   V   L  (SEQ ID NO: 55) 316AAG CTG ACC GTC CTA (SEQ ID NO: 56)

1.139.15 hAb-VH(23521-VH) (SEQ ID NO:57 for amino acid and SEQ ID NO:58for nucleic acid) with heavy chain CDRs 1-3: SEQ ID NOs: 21, 23, 25 areamino acid sequences and SEQ ID NO:22, 24, 26 are nucleic acidsequences, respectively:

V segment: IGHV4-39*01 D segment: IGHD6-13*01 J segment: IGHJ4*02 Q   L   Q   L   Q   E   S   G   P   G   L   V   K   P   S   1CAG CTG CAG CTG CAG GAG TCG GGC CCA GGA CTG GTG AAG CCC TCG E   T   L   S   L   T   C   T   V   S   G   G   S   I   S  46GAG ACC CTG TCC CTC ACC TGC ACT GTC TCT GGT GGC TCC ATC AGC            CDR1  ------------------------- S   T   T   Y   Y   W   G   W   I   R   Q   P   P   G   K  91AGT ACT ACT TAC TAC TGG GGC TGG ATC CGC CAG CCC CCA GGG AAG                                        CDR2                         ---------------------------------  G   L   E   W   I   G   S   I   S   Y   S   G   T   T   Y 136GGG CTG GAG TGG ATT GGG AGT ATC TCT TAT AGT GGG ACC ACC TAC            CDR2  ------------------------- Y   N   P   S   L   K   S   R   V   T   I   P   V   D   T 181TAC AAC CCG TCC CTC AAG AGT CGA GTC ACC ATC CCC GTA GAC ACG S   K   N   Q   I   S   L   K   L   S   S   V   T   A   A 226TCC AAG AAC CAG ATC TCC CTG AAA CTG AGC TCT GTG ACC GCC GCA                                                CDR3                                     --------------------- D   T   S   L   Y   Y   C   A   R   H   L   G   Y   N   S 261GAC ACG TCT TTG TAT TAT TGT GCG AGA CAT CTC GGG TAT AAC AGC            CDR3  ------------------------- N   W   Y   P   F   D   Y   W   G   Q   G   T   L   V   T 316AAC TGG TAC CCT TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC V   S   S  (SEQ ID NO: 57) 361 GTC TCC TCA (SEQ ID NO: 58)

1.139.15 hAb-VL(22895-VL) (SEQ ID NO:59 for amino acid and SEQ ID NO:60for nucleic acid) with light chain CDRs 1-3: SEQ ID NOs: 27, 29, 31 areamino acid sequences and SEQ ID NO: 28, 30, 32 are nucleic acidsequences, respectively:

V segment: IGLV2-18*02 J segment: IGLJ3*02 Q   S   A   L   T   Q   P   P   S   V   S   G   S   P   G   1CAG TCG GCC CTG ACT CAG CCT CCC TCC GTG TCC GGG TCT CCT GGA                                  CDR1                             ----------------------------- Q   S   V   T   I   S   C   T   G   T   S   S   D   V   G  46CAG TCA GTC ACC ATC TCC TGC ACT GGA ACC AGC AGT GAC GTT GGT         CDR1  --------------------- S   Y   N   R   V   S   W   Y   Q   Q   P   P   G   T   A  91AGT TAT AAC CGT GTC TCC TGG TAC CAG CAG CCC CCA GGC ACA GCC                                   CDR2                         ------------------------- P   E   V   I   I   Y   E   V   S   N   R   P   S   G   V 136CCC GAA GTC ATT ATT TAT GAG GTC AGT AAT CGG CCC TCA GGG GTC P   D   R   F   S   G   S   K   S   G   N   T   A   S   L 181CCT GAT CGC TTC TCT GGG TCC AAG TCT GGC AAC ACG GCC TCC CTG T   I   S   G   L   Q   A   E   D   E   A   D   Y   Y   C 226ACC ATC TCT GGG CTC CAG GCT GAG GAC GAG GCT GAT TAT TAC TGC                 CDR3  ------------------------------------- S   S   Y   T   S   S   S   T   W   V   F   G   G   G   T 261AGC TCA TAT ACA AGC AGC AGC ACT TGG GTG TTC GGC GGA GGG ACC K   L   T   V   L  (SEQ ID NO: 59) 316 AAG CTG ACC GTC CTA (SEQ ID NO: 60)

1.153.7 hAb-VH(20942-VH): (SEQ ID NO:61 for amino acid and SEQ ID NO:62for nucleic acid) with heavy chain CDRs 1-3: SEQ ID NOs: 33, 35, 37 areamino acid sequences and SEQ ID NO: 34, 36, 38 are nucleic acidsequences, respectively:

V segment: IGHV3-23*01 D segment: IGHD7-27*01 J segment: IGHJ4*02 E   V   Q   L   L   E   S   G   G   G   L   V   Q   P   G   1GAG GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTA CAG CCT GGG G   S   L   R   L   S   C   A   A   S   G   F   T   F   S  46GGG TCC CTG AGA CTG TCC TGC GCA GCC TCT GGA TTC ACC TTT AGC         CDR1 ----------------- S   H   A   M   S   W   V   R   Q   A   P   G   K   G   L  91AGC CAT GCC ATG AGC TGG GTC CGC CAG GCT CCA GGG AAG GGG CTG                                    CDR2                 ----------------------------------------- E   W   V   S   T   I   T   G   G   G   G   S   I   Y   Y 136GAG TGG GTC TCA ACT ATT ACT GGT GGT GGT GGT AGC ATA TAC TAC         CDR2 --------------------- A   D   S   V   K   G   R   F   T   I   S   R   D   N   S 181GCA GAC TCC GTG AAG GGC CGG TTC ACC ATC TCC AGA GAC AAT TCC K   N   T   L   Y   L   Q   M   N   S   L   R   A   E   D 226AAG AAC ACG CTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAG GAC                                            CDR3                                 ------------------------- T   A   V   Y   Y   C   A   K   N   R   A   G   E   G   Y 261ACG GCC GTA TAT TAT TGT GCG AAA AAC CGC GCT GGG GAG GGT TAC     CDR3 --------- F   D   Y   W   G   Q   G   T   L   V   T   V   S   S  (SEQ ID NO: 61)316TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA (SEQ ID NO: 62)

1.153.7 hAb-VL(21110-VL) (SEQ ID NO:63 for amino acid and SEQ ID NO:64for nucleic acid) with light chain CDRs 1-3: SEQ ID NOs: 39, 41, 43 areamino acid sequences and SEQ ID NO: 40, 42, 44 are nucleic acidsequences, respectively:

V segment: IGLV3-9*01 J segment: IGLJ3*02 S   Y   E   L   T   Q   P   L   S   V   S   V   A   L   G   1TCC TAT GAG CTG ACT CAG CCA CTC TCA GTG TCA GTG GCC CTG GGA                                        CDR1                             ----------------------------- Q   T   A   R   I   T   C   G   G   D   N   I   G   N   K  46CAG ACG GCC AGG ATT ACC TGT GGG GGA GAC AAC ATT GGA AAT AAA     CDR1 ---------  D   V   H   W   Y   Q   Q   K   P   G   Q   A   P   V   L 91 GAT GTG CAC TGG TAC CAG CAG AAG CCA GGC CAG GCC CCT GTG CTG                       CDR2              ------------------------ V   I   Y   R   D   S   N   R   P   S   G   I   P   E   G 136GTC ATC TAT AGG GAT AGC AAC CGG CCC TCT GGG ATC CCT GAG GGA F   S   G   S   N   S   G   N   T   A   T   L   T   I   S 181TTC TCT GGC TCC AAC TCG GGG AAC ACG GCC ACC CTG ACC ATC AGC                                                    CDR3                                                 --------- R   A   Q   A   G   D   E   A   D   Y   Y   C   Q   V   W 226AGA GCC CAA GCC GGG GAT GAG GCT GAC TAT TAC TGT CAG GTG TGG        CDR3 ----------------- D   S   I   W   V   F   G   G   G   T   K   L   T   V   L  (SEQ ID NO: 63)261GAC AGC ATT TGG GTG TTC GGC GGA GGG ACC AAG CTG ACC GTC CTA (SEQ ID NO: 64)

1.103.11-v2 hAb-VH(20975-VH): (SEQ ID NO:53 for amino acid and SEQ IDNO:54 for nucleic acid) with heavy chain CDRs 1-3: SEQ ID NOs: 1, 15, 5are amino acid sequences and SEQ ID NO:2, 16, 6 are nucleic acidsequences, respectively:

V segment: IGHV4-39*01 D segment: IGHD1-26*01 J segment: IGHJ4*02 Q   L   Q   L   Q   E   S   G   P   G   L   V   K   P   S   1CAG CTG CAG CTG CAG GAG TCG GGC CCA GGA CTG GTG AAG CCT TCG E   T   L   T   L   T   C   T   V   S   A   D   S   I   S  46GAG ACC CTG ACC CTC ACC TGC ACT GTC TCT GCT GAC TCC ATC AGC            CDR1  ------------------------- S   T   T   Y   Y   W   V   W   I   R   Q   P   P   G   K  91AGT ACT ACT TAC TAC TGG GTC TGG ATC CGC CAG CCC CCA GGG AAG                                        CDR2                         --------------------------------- G   L   E   W   I   G   S   I   S   Y   S   G   S   T   Y 136GGA CTG GAG TGG ATT GGG AGT ATC TCT TAT AGT GGG AGC ACC TAC            CDR2  ------------------------- Y   N   P   S   L   K   S   R   V   T   V   S   V   D   T 181TAC AAT CCG TCC CTC AAG AGT CGA GTC ACC GTA TCC GTA GAC ACG S   K   N   Q   F   S   L   K   L   N   S   V   A   A   T 226TCC AAG AAC CAG TTC TCC CTG AAG CTG AAC TCT GTG GCC GCC ACA                                            CDR3                                     --------------------- D   T   A   L   Y   Y   C   A   R   H   L   G   Y   N   G 261GAC ACG GCT CTA TAT TAC TGT GCG AGA CAT CTA GGG TAT AAT GGG            CDR3  ------------------------- R   Y   L   P   F   D   Y   W   G   Q   G   T   L   V   T 316AGG TAC CTC CCC TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC V   S   S  (SEQ ID NO: 53) 361 GTC TCC TCC (SEQ ID NO: 54)

1.103.11-v2 hAb-VL(21038-2-VL): (SEQ ID NO:67 for amino acid and SEQ IDNO:68 for nucleic acid) with light chain CDRs 1-3: SEQ ID NOs: 7, 17, 65are amino acid sequences and SEQ ID NO:8, 18, 66 are nucleic acidsequences, respectively:

V segment: IGLV2-14*01 J segment: IGLJ3*02 Q   S   A   L   T   Q   P   A   S   V   S   G   S   P   G   1CAG TCT GCC CTG ACT CAG CCT GCC TCC GTG TCT GGG TCT CCT GGA                                        CDR1                             ----------------------------- Q   S   I   T   I   S   C   T   G   T   S   S   D   V   G  46CAG TCG ATC ACC ATC TCC TGC ACT GGA ACC AGC AGT GAC GTT GGT         CDR1 --------------------- F   Y   N   Y   V   S   W   Y   Q   Q   H   P   G   K   A  91TTT TAT AAC TAT GTC TCC TGG TAC CAA CAG CAC CCA GGC AAA GCC                                    CDR2                         ------------------------- P   E   L   M   I   Y   D   V   S   N   R   P   S   G   V 136CCC GAA CTC ATG ATT TAT GAT GTC AGT AAT CGG CCC TCA GGG GTT S   D   R   F   S   G   S   K   S   G   N   T   A   S   L 181TCT GAT CGC TTC TCT GGC TCC AAG TCT GGC AAC ACG GCC TCC CTG T   I   S   G   L   Q   A   E   D   E   A   D   Y   Y   C 226ACC ATC TCT GGG CTC CAG GCT GAG GAC GAG GCT GAT TAT TAC TGC                CDR3  ------------------------------------- S   S   Y   T   S   I   S   T   W   V   F   G   G   G   T 261AGC TCA TAT ACA AGC ATC AGC ACT TGG GTG TTC GGC GGA GGG ACC K   L   T   V   L  (SEQ ID NO: 67) 316AAG CTG ACC GTC CTA (SEQ ID NO: 68)

In some embodiments, the anti-PD-1 antibodies and the antigen-bindingfragments thereof comprise a heavy chain CDR sequences selected from thegroup consisting of: SEQ ID NOs: 1, 3, 5, 13, 15, 21, 23, 25, 33, 35 and37. In some embodiments, the anti-PD-1 antibodies and theantigen-binding fragments thereof comprise a light chain CDR sequencesselected from the group consisting of: SEQ ID NOs: 7, 9, 11, 17, 19, 27,29, 31, 39, 41, 43 and 65. In certain embodiments, one or more CDRsequences provided herein can be modified or changed such that theresulting antibody is improved over the parent antibody in one or moreproperties (such as improved antigen-binding, improved glycosylationpattern, reduced risk of glycosylation on a CDR residue, reduceddeamination on a CDR residue, increased pharmacokinetic half-life, pHsensitivity, and compatibility to conjugation), and is otherwisecomparable to the parent antibody (i.e. antibody having otherwise thesame set of CDR sequences except for the above-mentioned modification orchange), or at least substantially retains the antigen-binding propertyof the parent antibody.

In some embodiments, the anti-PD-1 antibodies and the antigen-bindingfragments thereof comprise a heavy chain variable region selected fromthe group consisting of: a heavy chain variable region comprising SEQ IDNO: 1, SEQ ID NO: 3, and/or SEQ ID NO: 5; a heavy chain variable regioncomprising SEQ ID NO: 13, SEQ ID NO: 15, and/or SEQ ID NO: 5; a heavychain variable region comprising SEQ ID NO: 1, SEQ ID NO: 15, and/or SEQID NO: 5; a heavy chain variable region comprising SEQ ID NO: 21, SEQ IDNO: 23, and/or SEQ ID NO: 25; and a heavy chain variable regioncomprising SEQ ID NO: 33, SEQ ID NO: 35, and/or SEQ ID NO: 37.

In some embodiments, the anti-PD-1 antibodies and the antigen-bindingfragments thereof comprise a light chain variable region selected fromthe group consisting of: a light chain variable region comprising SEQ IDNO: 7, SEQ ID NO: 9, and/or SEQ ID NO: 11; a light chain variable regioncomprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 11; a lightchain variable region comprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQID NO: 19; a light chain variable region comprising SEQ ID NO: 27, SEQID NO: 29, and/or SEQ ID NO: 31; a light chain variable regioncomprising SEQ ID NO: 39, SEQ ID NO: 41, and/or SEQ ID NO: 43; and alight chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 17,and/or SEQ ID NO: 65.

In some embodiments, the anti-PD-1 antibodies and the antigen-bindingfragments thereof comprising: a) a heavy chain variable regioncomprising SEQ ID NO: 1, SEQ ID NO: 3, and/or SEQ ID NO: 5; and a lightchain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9, and/or SEQID NO: 11; b) a heavy chain variable region comprising SEQ ID NO: 13,SEQ ID NO: 15, and/or SEQ ID NO: 5; and a light chain variable regioncomprising SEQ ID NO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 11; c) a heavychain variable region comprising SEQ ID NO: 1, SEQ ID NO: 15, and/or SEQID NO: 5; and a light chain variable region comprising SEQ ID NO: 7, SEQID NO: 17, and/or SEQ ID NO: 19; d) a heavy chain variable regioncomprising SEQ ID NO: 21, SEQ ID NO: 23, and/or SEQ ID NO: 25 and alight chain variable region comprising SEQ ID NO: 27, SEQ ID NO: 29,and/or SEQ ID NO: 31; e) a heavy chain variable region comprising SEQ IDNO: 33, SEQ ID NO: 35, and/or SEQ ID NO: 37; and a light chain variableregion comprising SEQ ID NO: 39, SEQ ID NO: 41, and/or SEQ ID NO: 43; orf) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 15,and/or SEQ ID NO: 5; and a light chain variable region comprising SEQ IDNO: 7, SEQ ID NO: 17, and/or SEQ ID NO: 65.

A skilled artisan will understand that the CDR sequences provided inTable 1 can be modified to contain one or more substitutions of aminoacids, so as to provide for an improved biological activity such asimproved binding affinity to human PD-1. For example, a library ofantibody variants (such as Fab or scFv variants) can be generated andexpressed with phage display technology, and then screened for thebinding affinity to human PD-1. For another example, computer softwarecan be used to virtually simulate the binding of the antibodies to humanPD-1, and identify the amino acid residues on the antibodies which formthe binding interface. Such residues may be either avoided in thesubstitution so as to prevent reduction in binding affinity, or targetedfor substitution to provide for a stronger binding. In certainembodiments, at least one (or all) of the substitution(s) in the CDRsequences is conservative substitution.

In certain embodiments, the antibodies and the antigen-binding fragmentsthereof comprise one or more CDR sequences having at least 80% (e.g. atleast 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%)sequence identity to that (or those) listed in Table 1, and in themeantime retain the binding affinity to human PD-1 at a level similar toor even higher than its parental antibody having substantially the samesequence except that the corresponding CDR sequence is in 100% sequenceidentity to that (or those) listed in Table 1.

In certain embodiments, the anti-PD-1 antibodies and the antigen-bindingfragments thereof are fully human. The fully human antibodies do nothave the issues of immunogenicity in human and/or reduced bindingaffinity as often observed with humanized antibodies.

In some embodiments, the fully human anti-PD-1 antibodies and theantigen-binding fragments thereof comprise a heavy chain variable regionselected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 49, SEQID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, and a homologous sequencethereof having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%) sequence identity; and/or a light chainvariable region selected from the group consisting of: SEQ ID NO: 47,SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO:67, and a homologous sequence thereof having at least 80% (e.g. at least85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequenceidentity. Theses fully human antibodies retain the binding affinity tohuman PD-1, preferably at a level similar to one of the exemplaryantibodies: 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.103.11-v2 hAb,1.139.15 hAb, and 1.153.7 hAb.

In some embodiments, the fully human anti-PD-1 antibodies and theantigen-binding fragments thereof comprise: a) a heavy chain variableregion comprising SEQ ID NO: 45; and a light chain variable regioncomprising SEQ ID NO: 47; b) a heavy chain variable region comprisingSEQ ID NO: 49; and a light chain variable region comprising SEQ ID NO:51; c) a heavy chain variable region comprising SEQ ID NO: 53; and alight chain variable region comprising SEQ ID NO: 55; d) a heavy chainvariable region comprising SEQ ID NO: 57; and a light chain variableregion comprising SEQ ID NO: 59; e) a heavy chain variable regioncomprising SEQ ID NO: 61; and a light chain variable region comprisingSEQ ID NO: 63; or f) a heavy chain variable region comprising SEQ ID NO:53; and a light chain variable region comprising SEQ ID NO: 67.

Also contemplated herein are antibodies and the antigen-bindingfragments that compete for the same epitope with the anti-PD-1antibodies and the antigen-binding fragments thereof provided herein. Incertain embodiments, the antibodies block binding of 1.7.3 hAb, 1.49.9hAb, 1.103.11 hAb, 1.103.11-v2 hAb, 1.139.15 hAb, or 1.153.7 hAb tohuman or monkey PD-1, for example, at an IC₅₀ value (i.e. 50% inhibitionconcentration) of below 10⁻⁶ M, below 10⁻⁷ M, below 10^(−7.5) M, below10⁻⁸ M, below 10^(−8.5) M, below 10⁻⁹ M, or below 10⁻¹⁰ M. The IC₅₀values are determined based on a competition assay such as ELISA assays,radioligand competition binding assays, and FACS analysis.

In some embodiments, the anti-PD-1 antibodies and the antigen-bindingfragments thereof provided herein are capable of specifically binding tohuman PD-1 with a binding affinity (Kd) of ≤10⁻⁶ M (e.g., ≤5×10⁻⁷ M,≤2×10⁻⁷ M, ≤10⁻⁷ M, ≤5×10⁻⁸ M, ≤2×10⁻⁸ M, ≤10⁻⁸ M, ≤5×10⁻⁹ M, ≤2×10⁻⁹M,≤10⁻⁹ M, 10⁻¹⁰ M) as measured by plasmon resonance binding assay. Thebinding affinity can be represented by K_(D) value, which is calculatedas the ratio of dissociation rate to association rate (k_(off)/k_(on))when the binding between the antigen and the antigen-binding moleculereaches equilibrium. The antigen-binding affinity (e.g. K_(D)) can beappropriately determined using suitable methods known in the art,including, for example, plasmon resonance binding assay usinginstruments such as Biacore (see, for example, Murphy, M. et al, Currentprotocols in protein science, Chapter 19, unit 19.14, 2006).

In certain embodiments, the antibodies and the fragments thereofprovided herein binds to human PD-1 with an EC₅₀ (i.e. 50% bindingconcentration) of 0.1 nM-100 nM (e.g. 0.1 nM-50 nM, 0.1 nM-30 nM, 0.1nM-20 nM, 0.1 nM-10 nM, or 0.1 nM-1 nM). Binding of the antibodies tohuman PD-1 can be measured by methods known in the art, for example,sandwich assay such as ELISA, Western Blot, FACS or other binding assay.In an illustrative example, the test antibody (i.e. first antibody) isallowed to bind to immobilized human PD-1 or cells expressing humanPD-1, after washing away the unbound antibody, a labeled secondaryantibody is introduced which can bind to and thus allow detection of thebound first antibody. The detection can be conducted with a microplatereader when immobilized PD-1 is used, or by using FACS analysis whencells expressing human PD-1 are used. In certain embodiments, theantibodies and the fragments thereof provided herein binds to human PD-1with an EC₅₀ (i.e. 50% effective concentration) of 1 nM to 10 nM, or 1nM to 5 nM as measured by FACS analysis.

In certain embodiments, the antibodies and the fragments thereofprovided herein inhibit the binding of human PD-1 to its ligand at anIC₅₀ of 0.2 nM-100 nM (e.g. 0.2 nM-50 nM, 0.2 nM-30 nM, 0.2 nM-20 nM,0.2 nM-10 nM, or 1 nM-10 nM), as measured in a competition assay.

In certain embodiments, the antibodies and the fragments thereofprovided herein block binding of human PD-1 to its ligand and therebyproviding biological activity including, for example, inducing cytokineproduction from the activated T cells (such as CD4⁺ T cells and CD8⁺ Tcells), inducing proliferation of activated T cells (such as CD4+ Tcells and CD8⁺ T cells), and reversing T reg's suppressive function.Exemplary cytokines include IL-2 and IFNγ. The term “IL-2” refers tointerleukin 2, a type of cytokine signaling molecule in the immunesystem that regulates the activities of white blood cells (e.g.leukocytes). The term “Interferon gamma (IFNγ)” is a cytokine that isproduced by natural killer (NK), NK T cells, CD4⁺ and CD8⁺T cells, whichis a critical activator of macrophages and inducer of majorhistocompatibility complex (MHC) molecule expression. The cytokineproduction can be determined using methods known in the art, forexample, by ELISA. Methods can also be used to detect proliferation of Tcells, including [³H] thymidine incorporation assay.

The anti-PD-1 antibodies and the antigen-binding fragments thereof arespecific for PD-1. In certain embodiments, the antibodies andantigen-binding fragments thereof do not bind to CD28 and/or CTLA-4. Forexample, the binding affinity with CD28 and/or CTLA-4 is less than 15%,10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that with PD-1.

In certain embodiments, the antibodies and antigen-binding fragmentsthereof bind to monkey PD-1 at an EC50 of no more than 100 nM, forexample, no more than or about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM,0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM,0.04 nM, 0.03 nM, 0.02 nM, or 0.01 nM, as measured by ELISA. In certainembodiments, the antibodies and antigen-binding fragments thereof bindto monkey PD-1 at an EC50 of about 1 nM-10 nM.

In certain embodiments, the antibodies and antigen-binding fragmentsthereof do not bind to mouse PD-1 but bind to monkey PD-1 with a bindingaffinity similar to that of human PD-1. For example, binding of theexemplary antibodies 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.103.11-v2hAb, 1.139.15 hAb, and 1.153.7 hAb to mouse PD-1 is not detectable inconventional binding assays such as ELISA, or FACS analysis, whereas thebinding of these antibodies to monkey PD-1 is at a similar affinity orEC50 value to that of human PD-1 as measured by ELISA or FACS.

In some embodiments, the anti-PD-1 antibodies and the antigen-bindingfragments thereof has reduced or depleted effector function. In someembodiments, the anti-PD-1 antibodies and the antigen-binding fragmentsthereof have a constant region of IgG4 isotype, which has reduced ordepleted effector function. Effector functions such as ADCC and CDC canlead to cytotoxicity to cells expressing PD-1. Many cells such as Tcells normally express PD-1. In order to avoid potential unwantedtoxicity to those normal cells, certain embodiments of the antibodiesand antigen-binding fragments provided herein can possess reduced oreven depleted effector functions. Various assays are known to evaluateADCC or CDC activities, for example, Fc receptor binding assay, C1qbinding assay, and cell lysis assay, and can be readily selected bypeople in the art. Without wishing to be bound to theory, but it isbelieved that antibodies with reduced or depleted effector functionssuch as ADCC or CDC would cause no or minimal cytotoxicity toPD-1-expressing cells, for example those T cells, and therefore sparethem from unwanted side effects, whereas in the meantime, blocking ofPD-1 would boost immune system for the treatment of conditions such ascancer or chronic infection.

In certain embodiments, the anti-PD-1 antibodies and antigen-bindingfragments thereof provided herein have reduced side effects. Forexample, the antibodies and antigen-binding fragments thereof providedherein can have fully human IgG sequence and therefore reducedimmunogenicity than a humanized antibody counterpart. For anotherexample, the antibodies and antigen-binding fragments thereof providedherein can be in IgG4 format to eliminate ADCC and CDC.

In certain embodiments, the anti-PD-1 antibodies and antigen-bindingfragments thereof provided herein are advantageous in that they can beused in combination with immunogenic agents, such as tumor cells,purified tumor antigen, and cells transfected with genes encoding immunestimulating cytokines, tumor vaccines. In addition, the anti-PD-1antibodies and antigen-binding fragments thereof can be included incombination therapies, including standard chemo- and radio-therapies,target based small molecule therapies, emerging other immune checkpointmodulator therapies. In certain embodiments, the antibodies andantigen-binding fragments thereof can be used as the base ofantibody-drug conjugates, bispecific or multivalent antibodies.

The anti-PD-1 antibodies or antigen-binding fragments thereof providedherein can be a monoclonal antibody, polyclonal antibody, fully humanantibody, humanized antibody, chimeric antibody, recombinant antibody,bispecific antibody, labeled antibody, bivalent antibody, oranti-idiotypic antibody. A recombinant antibody is an antibody preparedin vitro using recombinant methods rather than in animals. A bispecificor bivalent antibody is an artificial antibody having fragments of twodifferent monoclonal antibodies and can bind two different antigens. Anantibody or antigen-binding fragment thereof that is “bivalent”comprises two antigen-binding sites. The two antigen binding sites maybind to the same antigen, or they may each bind to a different antigen,in which case the antibody or antigen-binding fragment is characterizedas “bispecific.”

In some embodiments, the anti-PD-1 antibodies or antigen-bindingfragments thereof provided herein are fully human antibodies. In certainembodiments, the fully human antibodies are prepared using recombinantmethods. For example, transgenic animal such as a mouse can be made tocarry transgenes or transchromosomes of human immunoglobulin genes, andtherefore capable of producing fully human antibodies after immunizationwith proper antigen such as human PD-1. Fully human antibodies can beisolated from such transgenic animal, or alternatively, can be made byhybridoma technology by fusing the spleen cells of the transgenic animalwith an immortal cell line to generate hybridoma cells secreting thefully human antibodies. Exemplary transgenic animals include, withoutlimitation, OmniRat, whose endogenous expression of rat immunoglobulingenes are inactivated and at the same time engineered to containfunctional recombinant human immunoglobulin loci; OmniMouse, whoseendogenous expression of mouse immunoglobulin genes are inactivated andat the same time engineered to contain recombinant human immunoglobulinloci having J-locus deletion and a C-kappa mutation; OmniFlic, which isa transgenic rat whose endogenous expression of rat immunoglobulin genesare inactivated and at the same time engineered to contain recombinanthuman immunoglobulin loci having a single common, rearranged VkJk lightchain and functional heavy chain. Detailed information can be furtherfound at: Osborn M. et al, Journal of Immunology, 2013, 190: 1481-90; MaB. et al, Journal of Immunological Methods 400-401(2013) 78-86; GeurtsA. et al, Science, 2009, 325:433; U.S. Pat. No. 8,907,157; EP patent2152880B1; EP patent 2336329B1, all of which are incorporated herein byreference to its entirety. Other suitable transgenic animals can also beused, for example, HuMab mice (see, for details, Lonberg, N. et al.Nature 368(6474): 856 859 (1994)), Xeno-Mouse (Mendez et al. Nat Genet.,1997, 15:146-156), TransChromo Mouse (Ishida et al. Cloning Stem Cells,2002, 4:91-102) and Veloclmmune Mouse (Murphy et al. Proc Natl Acad SciUSA, 2014, 111:5153-5158), Kymouse (Lee et al. Nat Biotechnol, 2014,32:356-363), and transgenic rabbit (Flisikowska et al. PLoS One, 2011,6:e21045).

In some embodiments, the anti-PD-1 antibodies and the antigen-bindingfragments thereof is a camelized single domain antibody, a diabody, ascFv, an scFv dimer, a BsFv, a dsFv, a (dsFv)2, a dsFv-dsFv′, an Fvfragment, a Fab, a Fab′, a F(ab′)2, a ds diabody, a nanobody, a domainantibody, or a bivalent domain antibody.

In some embodiments, the anti-PD-1 antibodies and the antigen-bindingfragments thereof further comprise an immunoglobulin constant region. Insome embodiments, an immunoglobulin constant region comprises a heavychain and/or a light chain constant region. The heavy chain constantregion comprises CH1, CH1-CH2, or CH1-CH3 regions. In some embodiments,the constant region may further comprise one or more modifications toconfer desirable properties. For example, the constant region may bemodified to reduce or deplete one or more effector functions, to improveFcRn receptor binding, or to introduce one or more cysteine residues.

In some embodiments, the anti-PD-1 antibodies and the antigen-bindingfragments thereof further comprise a conjugate. It is contemplated thata variety of conjugates may be linked to the antibodies orantigen-binding fragments provided herein (see, for example, “ConjugateVaccines”, Contributions to Microbiology and Immunology, J. M. Cruse andR. E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). Theseconjugates may be linked to the antibodies or antigen-binding fragmentsby covalent binding, affinity binding, intercalation, coordinatebinding, complexation, association, blending, or addition, among othermethods. In certain embodiments, the antibodies and antigen-bindingfragments disclosed herein may be engineered to contain specific sitesoutside the epitope binding portion that may be utilized for binding toone or more conjugates. For example, such a site may include one or morereactive amino acid residues, such as for example cysteine or histidineresidues, to facilitate covalent linkage to a conjugate. In certainembodiments, the antibodies may be linked to a conjugate indirectly, orthrough another conjugate. For example, the antibody or antigen-bindingfragments may be conjugated to biotin, then indirectly conjugated to asecond conjugate that is conjugated to avidin. The conjugate can be adetectable label, a pharmacokinetic modifying moiety, a purificationmoiety, or a cytotoxic moiety. Examples of detectable label may includea fluorescent labels (e.g. fluorescein, rhodamine, dansyl,phycoerythrin, or Texas Red), enzyme-substrate labels (e.g. horseradishperoxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme,saccharide oxidases or β-D-galactosidase), radioisotopes (e.g. ¹²³I,¹²⁴I, ¹²⁵I, ¹³¹I, ³⁵S, ³H, ¹¹¹In, ¹¹²In, ¹⁴C, ⁶⁴Cu, ⁶⁷Cu, ⁸⁶Y, ⁸⁸Y, ⁹⁰Y,¹⁷⁷Lu, ²¹¹At, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, and ³²P, other lanthanides,luminescent labels), chromophoric moiety, digoxigenin, biotin/avidin, aDNA molecule or gold for detection. In certain embodiments, theconjugate can be a pharmacokinetic modifying moiety such as PEG whichhelps increase half-life of the antibody. Other suitable polymersinclude, such as, carboxymethylcellulose, dextran, polyvinyl alcohol,polyvinyl pyrrolidone, copolymers of ethylene glycol/propylene glycol,and the like. In certain embodiments, the conjugate can be apurification moiety such as a magnetic bead. A “cytotoxic moiety” can beany agent that is detrimental to cells or that can damage or kill cells.Examples of cytotoxic moiety include, without limitation, taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, puromycin and analogs thereof,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Polynucleotides and Recombinant Methods

The present disclosure provides isolated polynucleotides that encode theanti-PD-1 antibodies and the antigen-binding fragments thereof. Incertain embodiments, the isolated polynucleotides comprise one or morenucleotide sequences as shown in Table 1, which encodes the CDRsequences provided in Table 1.

In some embodiments, the isolated polynucleotides encodes a heavy chainvariable region and comprise a sequence selected from the groupconsisting of: SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO:58, SEQ ID NO: 62, and a homologous sequence thereof having at least 80%(e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%) sequence identity. In some embodiments, the isolatedpolynucleotides encodes a light chain variable region and comprise asequence selected from the group consisting of: SEQ ID NO: 48, SEQ IDNO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, anda homologous sequence thereof having at least 80% (e.g. at least 85%,88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequenceidentity. In certain embodiments, the percentage identity is due togenetic code degeneracy, while the encoded protein sequence remainsunchanged.

The isolated polynucleotide that encodes the anti-PD-1 antibodies andthe antigen-binding fragments thereof (e.g. including the sequences inTable 1) can be inserted into a vector for further cloning(amplification of the DNA) or for expression, using recombinanttechniques known in the art. In another embodiment, the antibody may beproduced by homologous recombination known in the art. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter (e.g. SV40, CMV, EF-1α), and atranscription termination sequence.

In some embodiments, the vector system includes mammalian, bacterial,yeast systems, etc, and comprises plasmids such as, but not limited to,pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP,pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO,Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2etc, and other laboratorial and commercially available vectors. Suitablevectors may include, plasmid, or viral vectors (e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses).

Vectors comprising the polynucleotide sequence encoding the antibody orantigen-binding fragment can be introduced to a host cell for cloning orgene expression. Suitable host cells for cloning or expressing the DNAin the vectors herein are the prokaryote, yeast, or higher eukaryotecells described above. Suitable prokaryotes for this purpose includeeubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as wellas Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such asP. aeruginosa, and Streptomyces.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-PD-1antibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibodies orantigen-fragment provided here are derived from multicellular organisms.Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruiffly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells. Plant cell cultures of cotton, corn, potato, soybean,petunia, tomato, and tobacco can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2). In some preferable embodiments, the host cell is 293F cell.

Host cells are transformed with the above-described expression orcloning vectors for anti-PD-1 antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

The host cells used to produce the antibodies or antigen-bindingfragments provided herein may be cultured in a variety of media.Commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium (DMEM), Sigma) are suitable for culturing thehost cells. In addition, any of the media described in Ham et al., Meth.Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culturemedia for the host cells. Any of these media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleotides (such as adenosine and thymidine), antibiotics (such asGENTAMYCIN™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody prepared from the cells can be purified using, for example,hydroxylapatite chromatography, gel electrophoresis, dialysis,DEAE-cellulose ion exchange chromatography, ammonium sulfateprecipitation, salting out, and affinity chromatography, with affinitychromatography being the preferred purification technique. Thesuitability of protein A as an affinity ligand depends on the speciesand isotype of any immunoglobulin Fc domain that is present in theantibody. Protein A can be used to purify antibodies that are based onhuman .gamma.1, .gamma.2, or .gamma.4 heavy chains (Lindmark et al., J.Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouseisotypes and for human .gamma.3 (Guss et al., EMBO J. 5:1567 1575(1986)). The matrix to which the affinity ligand is attached is mostoften agarose, but other matrices are available. Mechanically stablematrices such as controlled pore glass or poly(styrenedivinyl)benzeneallow for faster flow rates and shorter processing times than can beachieved with agarose. Where the antibody comprises a CH3 domain, theBakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) is useful forpurification. Other techniques for protein purification such asfractionation on an ion-exchange column, ethanol precipitation, ReversePhase HPLC, chromatography on silica, chromatography on heparinSEPHAROSE™ chromatography on an anion or cation exchange resin (such asa polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation are also available depending on the antibody to berecovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Kits

The present disclosure provides kits comprising the anti-PD-1 antibodiesor the antigen-binding fragments thereof. In some embodiments, the kitsare useful for detecting the presence or level of PD-1 in a biologicalsample. The biological sample can comprise a cell or a tissue.

In some embodiments, the kit comprises an anti-PD-1 antibody or theantigen-binding fragment thereof which is conjugated with a detectablelabel. In certain other embodiments, the kit comprises an unlabeledanti-PD-1 antibody or antigen-binding fragment, and further comprises asecondary labeled antibody which is capable of binding to the unlabeledanti-PD-1 antibody. The kit may further comprise an instruction of use,and a package that separates each of the components in the kit.

In certain embodiments, the anti-PD-1 antibody or the antigen-bindingfragment thereof are associated with a substrate or a device useful in asandwich assay such as ELISA, or in an immunographic assay. Usefulsubstrate or device can be, for example, microtiter plate and teststrip.

Pharmaceutical Composition and Method of Treatment

The present disclosure further provides pharmaceutical compositionscomprising the anti-PD-1 antibodies or the antigen-binding fragmentsthereof and one or more pharmaceutically acceptable carriers.

Pharmaceutical acceptable carriers for use in the pharmaceuticalcompositions disclosed herein may include, for example, pharmaceuticallyacceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueousvehicles, antimicrobial agents, isotonic agents, buffers, antioxidants,anesthetics, suspending/dispending agents, sequestering or chelatingagents, diluents, adjuvants, excipients, or non-toxic auxiliarysubstances, other components known in the art, or various combinationsthereof.

Suitable components may include, for example, antioxidants, fillers,binders, disintegrants, buffers, preservatives, lubricants, flavorings,thickeners, coloring agents, emulsifiers or stabilizers such as sugarsand cyclodextrins. Suitable antioxidants may include, for example,methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase,citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol,butylated hydroxanisol, butylated hydroxytoluene, and/or propyl gallate.As disclosed herein, inclusion of one or more antioxidants such asmethionine in a composition comprising an antibody or antigen-bindingfragment and conjugates as provided herein decreases oxidation of theantibody or antigen-binding fragment. This reduction in oxidationprevents or reduces loss of binding affinity, thereby improving antibodystability and maximizing shelf-life. Therefore, in certain embodimentscompositions are provided that comprise one or more antibodies orantigen-binding fragments as disclosed herein and one or moreantioxidants such as methionine. Further provided are methods forpreventing oxidation of, extending the shelf-life of, and/or improvingthe efficacy of an antibody or antigen-binding fragment as providedherein by mixing the antibody or antigen-binding fragment with one ormore antioxidants such as methionine.

To further illustrate, pharmaceutical acceptable carriers may include,for example, aqueous vehicles such as sodium chloride injection,Ringer's injection, isotonic dextrose injection, sterile waterinjection, or dextrose and lactated Ringer's injection, nonaqueousvehicles such as fixed oils of vegetable origin, cottonseed oil, cornoil, sesame oil, or peanut oil, antimicrobial agents at bacteriostaticor fungistatic concentrations, isotonic agents such as sodium chlorideor dextrose, buffers such as phosphate or citrate buffers, antioxidantssuch as sodium bisulfate, local anesthetics such as procainehydrochloride, suspending and dispersing agents such as sodiumcarboxymethylcelluose, hydroxypropyl methylcellulose, orpolyvinylpyrrolidone, emulsifying agents such as Polysorbate 80(TWEEN-80), sequestering or chelating agents such as EDTA(ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraaceticacid), ethyl alcohol, polyethylene glycol, propylene glycol, sodiumhydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobialagents utilized as carriers may be added to pharmaceutical compositionsin multiple-dose containers that include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Suitable excipients may include, for example, water, saline, dextrose,glycerol, or ethanol. Suitable non-toxic auxiliary substances mayinclude, for example, wetting or emulsifying agents, pH bufferingagents, stabilizers, solubility enhancers, or agents such as sodiumacetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

The pharmaceutical compositions can be a liquid solution, suspension,emulsion, pill, capsule, tablet, sustained release formulation, orpowder. Oral formulations can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,polyvinyl pyrollidone, sodium saccharine, cellulose, magnesiumcarbonate, etc.

In embodiments, the pharmaceutical compositions are formulated into aninjectable composition. The injectable pharmaceutical compositions maybe prepared in any conventional form, such as for example liquidsolution, suspension, emulsion, or solid forms suitable for generatingliquid solution, suspension, or emulsion. Preparations for injection mayinclude sterile and/or non-pyretic solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent just prior to use, including hypodermic tablets,sterile suspensions ready for injection, sterile dry insoluble productsready to be combined with a vehicle just prior to use, and sterileand/or non-pyretic emulsions. The solutions may be either aqueous ornonaqueous.

In certain embodiments, unit-dose parenteral preparations are packagedin an ampoule, a vial or a syringe with a needle. All preparations forparenteral administration should be sterile and not pyretic, as is knownand practiced in the art.

In certain embodiments, a sterile, lyophilized powder is prepared bydissolving an antibody or antigen-binding fragment as disclosed hereinin a suitable solvent. The solvent may contain an excipient whichimproves the stability or other pharmacological components of the powderor reconstituted solution, prepared from the powder. Excipients that maybe used include, but are not limited to, water, dextrose, sorbital,fructose, corn syrup, xylitol, glycerin, glucose, sucrose or othersuitable agent. The solvent may contain a buffer, such as citrate,sodium or potassium phosphate or other such buffer known to those ofskill in the art at, in one embodiment, about neutral pH. Subsequentsterile filtration of the solution followed by lyophilization understandard conditions known to those of skill in the art provides adesirable formulation. In one embodiment, the resulting solution will beapportioned into vials for lyophilization. Each vial can contain asingle dosage or multiple dosages of the anti-PD-1 antibody orantigen-binding fragment thereof or composition thereof. Overfillingvials with a small amount above that needed for a dose or set of doses(e.g., about 10%) is acceptable so as to facilitate accurate samplewithdrawal and accurate dosing. The lyophilized powder can be storedunder appropriate conditions, such as at about 4° C. to roomtemperature.

Reconstitution of a lyophilized powder with water for injection providesa formulation for use in parenteral administration. In one embodiment,for reconstitution the sterile and/or non-pyretic water or other liquidsuitable carrier is added to lyophilized powder. The precise amountdepends upon the selected therapy being given, and can be empiricallydetermined.

Therapeutic methods are also provided, comprising: administering atherapeutically effective amount of the antibody or antigen-bindingfragment as provided herein to a subject in need thereof, therebytreating or preventing a condition or a disorder associated with relatedto PD-1. In another aspect, methods are provided to treat a condition ina subject that would benefit from upregulation of immune response,comprising administering a therapeutically effective amount of theantibody or antigen-binding fragment as provided herein to a subject inneed thereof.

The therapeutically effective amount of an antibody or antigen-bindingfragment as provided herein will depend on various factors known in theart, such as for example body weight, age, past medical history, presentmedications, state of health of the subject and potential forcross-reaction, allergies, sensitivities and adverse side-effects, aswell as the administration route and extent of tumor development.Dosages may be proportionally reduced or increased by one of ordinaryskill in the art (e.g., physician or veterinarian) as indicated by theseand other circumstances or requirements.

In certain embodiments, an antibody or antigen-binding fragment asprovided herein may be administered at a therapeutically effectivedosage of about 0.01 mg/kg to about 100 mg/kg (e.g., about 0.01 mg/kg,about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg,about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg,about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about100 mg/kg). In certain of these embodiments, the antibody orantigen-binding fragment is administered at a dosage of about 50 mg/kgor less, and in certain of these embodiments the dosage is 10 mg/kg orless, 5 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kgor less. In certain embodiments, the administration dosage may changeover the course of treatment. For example, in certain embodiments theinitial administration dosage may be higher than subsequentadministration dosages. In certain embodiments, the administrationdosage may vary over the course of treatment depending on the reactionof the subject.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single dose may beadministered, or several divided doses may be administered over time.

The antibodies and antigen-binding fragments disclosed herein may beadministered by any route known in the art, such as for exampleparenteral (e.g., subcutaneous, intraperitoneal, intravenous, includingintravenous infusion, intramuscular, or intradermal injection) ornon-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal,or topical) routes.

Conditions and disorders associated with PD-1 can be immune relateddisease or disorder. In certain embodiments, the PD-1 associatedconditions and disorders include tumors and cancers, for example,non-small cell lung cancer, small cell lung cancer, renal cell cancer,colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer,gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma,melanoma, head and neck cancer, thyroid cancer, sarcoma, prostatecancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia,lymphomas, myelomas, mycoses fungoids, merkel cell cancer, and otherhematologic malignancies, such as classical Hodgkin lymphoma (CHL),primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-celllymphoma, EBV-positive and -negative PTLD, and EBV-associated diffuselarge B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodalNK/T-cell lymphoma, nasopharyngeal carcinoma, and HHV8-associatedprimary effusion lymphoma, Hodgkin's lymphoma, neoplasm of the centralnervous system (CNS), such as primary CNS lymphoma, spinal axis tumor,brain stem glioma. In certain embodiments, the tumors and cancers aremetastatic, especially metastatic tumors expressing PD-L1. In certainembodiments, the PD-1 associated conditions and disorders includeautoimmune diseases, such as systemic lupus erythematosus (SLE),psoriasis, systemic scleroderma, autoimmune diabetes and the like. Incertain embodiments, the PD-1 associated conditions and disordersinclude infectious disease such as chronic viral infection, for example,viral infection of hepatitis B, hepatitis C, herpes virus, Epstein-Barrvirus, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplexvirus type 2, human papilloma virus, adenovirus, Kaposi West sarcomaassociated herpes virus epidemics, thin ring virus (Torquetenovirus), JCvirus or BK virus.

Methods of Use

The present disclosure further provides methods of using the anti-PD-1antibodies or the antigen-binding fragments thereof.

In some embodiments, the present disclosure provides methods of treatinga condition or a disorder associated with related to PD-1 in anindividual, comprising administering a therapeutically effective amountof the anti-PD-1 antibody or antigen-binding fragment thereof. Incertain embodiments, the individual has been identified as having adisorder or condition likely to respond to a PD-1 antagonist.

The presence or level of PD-L1 on an interested biological sample can beindicative of whether the individual from whom the biological sample isderived could likely respond to a PD-1 antagonist. Various methods canbe used to determine the presence or level of PD-L1 in a test biologicalsample from the individual. For example, the test biological sample canbe exposed to anti-PD-L1 antibody or antigen-binding fragment thereof,which binds to and detects the expressed PD-L1 protein. Alternatively,PD-L1 can also be detected at nucleic acid expression level, usingmethods such as qPCR, reverse transcriptase PCR, microarray, SAGE, FISH,and the like. In some embodiments, the test sample is derived from acancer cell or tissue, or tumor infiltrating immune cells. In certainembodiments, presence or upregulated level of the PD-L1 in the testbiological sample indicates likelihood of responsiveness. The term“upregulated” as used herein, refers to an overall increase of no lessthan 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80% or greater, in the protein level of PD-L1 in the test sample asdetected using the antibodies or antigen-binding fragments providedherein, as compared to the PD-L1 protein level in a reference sample asdetected using the same antibody. The reference sample can be a controlsample obtained from a healthy or non-diseased individual, or a healthyor non-diseased sample obtained from the same individual from whom thetest sample is obtained. For example, the reference sample can be anon-diseased sample adjacent to or in the neighborhood of the testsample (e.g. tumor).

The antibodies or antigen-binding fragments disclosed herein may beadministered alone or in combination with one or more additionaltherapeutic means or agents. For example, the antibodies orantigen-binding fragments disclosed herein may be administered incombination with chemotherapy, radiation therapy, surgery for thetreatment of cancer (e.g., tumorectomy), one or more anti-emetics orother treatments for complications arising from chemotherapy, or anyother therapeutic agent for use in the treatment of cancer or anymedical disorder mediated by PD-1. In certain of these embodiments, anantibody or antigen-binding fragment as disclosed herein that isadministered in combination with one or more additional therapeuticagents may be administered simultaneously with the one or moreadditional therapeutic agents, and in certain of these embodiments theantibody or antigen-binding fragment and the additional therapeuticagent(s) may be administered as part of the same pharmaceuticalcomposition. However, an antibody or antigen-binding fragmentadministered “in combination” with another therapeutic agent does nothave to be administered simultaneously with or in the same compositionas the agent. An antibody or antigen-binding fragment administered priorto or after another agent is considered to be administered “incombination” with that agent as the phrase is used herein, even if theantibody or antigen-binding fragment and second agent are administeredvia different routes. Where possible, additional therapeutic agentsadministered in combination with the antibodies or antigen-bindingfragments disclosed herein are administered according to the schedulelisted in the product information sheet of the additional therapeuticagent, or according to the Physicians' Desk Reference 2003 (Physicians'Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457;57th edition (November 2002)) or protocols well known in the art.

In certain embodiments, the therapeutic agents can induce or boostimmune response against cancer. For example, a tumor vaccine can be usedto induce immune response to certain tumor or cancer. Cytokine therapycan also be used to enhance tumor antigen presentation to the immunesystem. Examples of cytokine therapy include, without limitation,interferons such as interferon-α, -β, and -γ, colony stimulating factorssuch as macrophage-CSF, granulocyte macrophage CSF, and granulocyte-CSF,interleukins such IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, and IL-12, tumor necrosis factors such as TNF-α andTNF-β. Agents that inactivate immunosuppressive targets can also beused, for example, TGF-beta inhibitors, IL-10 inhibitors, and Fas ligandinhibitors. Another group of agents include those that activate immuneresponsiveness to tumor or cancer cells, for example, those enhance Tcell activation (e.g. agonist of T cell costimulatory molecules such asCTLA-4, ICOS and OX-40), and those enhance dendritic cell function andantigen presentation.

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. All specific compositions, materials, and methods describedbelow, in whole or in part, fall within the scope of the presentinvention. These specific compositions, materials, and methods are notintended to limit the invention, but merely to illustrate specificembodiments falling within the scope of the invention. One skilled inthe art may develop equivalent compositions, materials, and methodswithout the exercise of inventive capacity and without departing fromthe scope of the invention. It will be understood that many variationscan be made in the procedures herein described while still remainingwithin the bounds of the present invention. It is the intention of theinventors that such variations are included within the scope of theinvention.

Example 1: Antibody Hybridoma Generation

1.1 Immunogen generation: DNAs encoding PD-1 and PD-L1 ECD or fulllength were synthesized and inserted into the expression vectorpcDNA3.3. Max-prep the plasmid DNAs and the inserted DNA sequences wereverified by sequencing. Fusion proteins PD-1 ECD and PD-L1 ECDcontaining various tags, including human Fc, mouse Fc and His tags, wereobtained by transfection of human PD-1 ECD gene into CHO-S or HEK293cells. After 5 days, supernatants harvested from the culture oftransiently transfected cells were used for protein purification. Thefusion proteins were purified and quantitated for usage of immunizationand screening.

1.2 Stable cell lines establishment. In order to obtain tools forantibody screening and validation, we generated PD-1 and PD-L1transfectant cell lines. Briefly, CHO-K1, 293F or Ba/F3 cells weretransfected with pCND3.3 expression vector containing full-length PD-1or PD-L1 using Lipofectamine 2000 Transfection kit according tomanufacturer's protocol. At 48-72 hours post transfection, thetransfected cells were cultured in medium containing Blasticidin or G418for selection. Overtime this will select the cells that have stablyincorporated PD-1 or PD-L1 genes into their genomic DNAs. Meanwhile thecells were checked for interested genes PD-1 and PD-L1 expression. Oncethe expression verified, single clones of interested were picked up bylimited dilution and scaled up to large volumes. The establishedmonoclonal cell lines then were maintained in medium containing lowerdose of antibiotics Blasticidin or G418.

1.3 Antibody hybridoma generation.

1.3.1 Immunization and cell fusion: OMT-rats (obtained from OpenMonoclonal Technology, Inc., Palo Alto, US), 8-10 weeks of age, wereimmunized with 10 μg of human PD-1 ECD protein in TiterMax in footpadfor first boost, repeat the immunization every 3 days with PD-1 ECDprotein in Aluminium. Bleed rats every two weeks for serum collectionand antibody titers were measured by ELISA or FACS assay. When theantibody titer reached sufficient high, rats were given a final boostwithout adjuvant (add 100 μl 1×PBS instead) and cell fusion wasperformed as following: B lymphocytes isolated from lymph node ofimmunized OMT-rats were combined with myeloma cells (at 1:1 ratio). Cellmixture were washed and suspended with 5-10 ml ECF solution. Add ECFsolution to adjust the concentration to 2×10⁶ cells/ml. After electroniccell fusion, cell suspension from the fusion chamber was immediatelytransferred into a sterile tube containing more volume of medium. Afterincubation for more than 24 hours in a 37° C., the cell suspension wasmixed and pipetted into 96-well plates (0.5×10⁶ cells/plate). Cells wereincubated at 37° C., 5% CO₂. When the clones were big enough, transfer100 μl supernatant from the 96-well plates to assay for antibodyscreening.

1.3.2 First and confirmation screen of hybridoma supernatants: ELISAassay was used as first screen method to test the binding of hybridomasupernatants to PD-1 protein. Briefly, Plates (Nunc) were coated withsoluble protein of human PD-1 extracellular domain at 1 μg/ml overnightat 4° C. After blocking and washing, the hybridoma supernatants weretransferred to the coated plates and incubate at room temperature for 1h. The plates were then washed and subsequently incubated with secondaryantibody goat anti rat IgG1 HRP (Bethyl) and goat anti rat IgG2b HRP(Bethyl) for 45 min. After washing, TMB substrate was added and theinteraction was stopped by 2M HCl. The absorbance at 450 nm was readusing a microplate reader (Molecular Device). In order to confirm thenative binding of PD-1 antibodies on conformational PD-1 moleculesexpressed on cell membrane, FACS analysis was performed on PD-1transfected CHO-S cell line. CHO-S cells expressing human PD-1 weretransferred in to 96-well U-bottom plates (BD) at a density of 1×10⁶cells/ml. The hybridoma supernatants were then transferred to the platesand incubated for 1 h at 4° C. After washing with 1×PBS/1% BSA, thesecondary antibody goat anti rat FITC (Jackson Immunoresearch Lab) wasapplied and incubated with cells at 4° C. in the dark for 1 h. The cellswere then washed and resuspended in 1×PBS/1% BSA or fixed with 4%paraformaldehyde, and analyzed by flow cytometery (BD). Antibody bindingto parental CHO-S cell line was performed using the same method. FIG. 1shows the binding of anti-human PD-1 antibodies to PD-1 expressing CHOcell. The CHO cells transfected with full-length human PD-1 were stainedwith antibodies against human PD-1 from rat hybridoma, followed by 2ndantibody staining with FITC conjugated goat anti-rat-lgG Fc and analyzedby FACS. The data show that the antibodies specifically bind to PD-1expressed on CHO cells.

To test the binding affinity of the antibodies to native PD-1 expressedon human CD4+T cells, human CD4+T cell were generated from PBMC culturedin IL-2 and OKT3 for 3 days and were stained with the antibodies againsthuman PD-1. Binding of the antibodies to the PD-1 on the T cells wereanalyzed by FACS. As shown in FIG. 3 , FACS analysis showed that theantibodies specially bind to native PD-1 expressed on CD4+T cells.

Testing the blocking activity of antibodies was used as confirmationscreen to select potential antibody hits. Selected antibodies weretested for the ability to block the binding of the ligand PD-L1 to PD-1transfected CHO-S cells by FACS analysis. CHO-S cells expressing humanPD-1 were transferred in to 96-well U-bottom plates (BD) at a density of1×10⁶ cells/ml. Antibodies were serially diluted in wash buffer(1×PBS/1% BSA) and incubated with the cells at 4° C. for 1 h. Afterwashing, human Fc fusion-human PD-L1 protein was added and incubated at4° C. for 1 h. The secondary antibody goat anti human IgG Fc FITCantibody (no cross-reactivity to rat IgG Fc, Jackson Immunoresearch Lab)was incubated with cells at 4° C. in the dark for 1 h. The cells werethen washed and resuspended in 1×PBS/1% BSA or fixed with 4%paraformaldehyde, and analyzed by flow cytometery (BD).

1.3.3 Hybridoma subcloning: once specific binding and blocking wereverified through first and confirmation screen, the positive hybridomacell lines can be used for subcloning. Briefly, for each hybridoma cellline, cells were counted and diluted to give 5 cells/well, 1 cell/welland 0.5 cell/well in cloning medium. Plate 200 μl/well into 96-wellplates, one plate at 5 cells/well, one plate at 1 cell/well and fourplates at 0.5 cell/well. Place all plates in incubator at 37° C., 5%CO₂. Incubate until all the cell lines can be checked by ELISA assay.

Example 2: Antibody Hybridoma Cell Sequence and Fully Human AntibodyCharacterization

2.1 Antibody hybridoma cell sequence: RNAs were isolated from monoclonalhybridoma cells with Trizol reagent. The VH and VL of PD-1 antibodieswere amplified as following protocol: briefly, RNA is first reversetranscribed into cDNA using a reverse transcriptase as described here,Reaction system (20 μl):

10 × RT Buffer 2.0 μl 25 × dNTP Mix (100 mM) 0.8 μl 10 × RT RandomPrimers/oligodT/specific primer 2.0 μ1 MμltiScribe ™ ReverseTranscriptase 1.0 μl RNase Inhibitor 1.0 μl RNA   2 μg Nuclease-free H₂Oto 20.0 μl

Reaction condition

Step1 Step 2 Step3 Step4 Temperature 25 37 85 4 Time 10 min 120 min 5min ∞

The resulting cDNA is used as templates for subsequent PCR amplificationusing primers specific for interested genes. The PCR reaction was doneas following procedure;

cDNA   1 μl Ex PCR buffer   5 μl dNTP   2 μl ExTaq  0.5 μl P1(25 pM) 0.5 μl P2(25 pM)  0.5 μl ddH₂O 40.5 μl

Reaction condition:

94° C.  3 min 94° C. 30 s 60° C. 30 s {close oversize brace} 30 cycles72° C.  1 min 72° C. 10 min

Take 10 μl of PCR reaction to do the ligation with pMD18-T vector. Dothe transformation of Top10 competent cells with 10 μl ligation productsand Transfer the mixture onto the pre-warmed 2-YT+Cab plates follow thestandard protocol, incubate overnight. Positive clones were checked byPCR using M13-48 and M13-47 primers followed by sequencing.

2.2 fully human antibody molecule construction: The VH and VL of PD-1antibodies were amplified as described above. The PCR reactions werepurified with PCR clean-up kit and the VL and pCI vector were digestedwith restriction enzymes Pme I and BssH II at 37° C. for 2 hours. Runthe reactions in 1% agarose and do gel extraction with kit according tomanufacturer's instruction. Ligation of digested VL and pCI vector asfollowing procedures:

Component Volume pCI-vector   80 ng VL fragments (insert)  100 ng T4 DNAligase Buffer   1 μl T4 DNA ligase  0.5 μl ddH₂O To 10 μl.

The mixture was incubated at 16° C. for 30 minutes. 10 μl of thereactions was used for transformation and clone growth. Confirmed cloneswere used for the extraction of the plasmid pCI-VL DNA. The pCI-VLvector and VH fragment were then digested with Xbal and Sal I and thepurified digested VH and vector were ligated with T4 DNA ligase 30minutes at 16° C. Once the sequence of inserted VL and VH were verifiedby sequencing, the expression vector containing whole IgG of fully humanPD-1 antibody was used for transient transfection and stable cell linedevelopment.

Example 3: Fully Human Antibody Characterization

3.1 Binding affinity of PD-1 antibodies to cell surface PD-1 moleculestested by flow cytometry (FACS): Antibody binding affinity to cellsurface PD-1 was performed by FACS analysis. CHO-S cells expressinghuman PD-1 were transferred in to 96-well U-bottom plates (BD) at adensity of 5×10⁵ cells/ml. Tested antibodies were 1:2 serially dilutedin wash buffer (1×PBS/1% BSA) and incubated with cells at 4° C. for 1 h.The secondary antibody goat anti-human IgG Fc FITC (3.0 moles FITC permole IgG, (Jackson Immunoresearch Lab) was added and incubated at 4° C.in the dark for 1 h. The cells were then washed once and resuspended in1×PBS/1% BSA, and analyzed by flow cytometery (BD). Fluorescenceintensity will be converted to bound molecules/cell based on thequantitative beads Quantum™ MESF Kits, Bangs Laboratories, Inc.). KD wascalculated using Graphpad Prism5. FIG. 2 shows the binding of the fullyhuman PD-1 antibodies (i.e. 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb,1.139.15 hAb, and 1.153.7 hAb) to PD-1 expressing CHO cell. Fully humanantibodies against human PD-1 were used to stain the PD-1 transfectedCHO cells and the FACS analysis show that fully human PD-1 antibodiesspecially bind to PD-1 with EC₅₀ about 2 nmol/L.

The 1.103.11-v2 hAb was generated by mutating a single amino acid Asn93(Kabat Numbering) on the original antibody 1.103.11 hAb-VH (20975-VL) toresidue Serine, so as to reduce the risk of glycosylation on the CDRresidue. Although Asn93 is located in light chain CDR3, theantibody-antigen complex model generated from computational dockingsuggested that Asn93 has no direct contact with any residue on theantigen of human PD-1. Most of the binding function of the light chainCDR3 seemed to be contributed by the neighboring residue Tyr91, whichhas interactions with some residues on PD-1 FG loop. The cell-basedfunctional assays of 1.103.11-v2 hAb confirmed that the mutation did notaffect any binding capability (see below experimental results and FIGS.15 and 16 ).

The binding affinity of 1.103.11-v2 hAb to human PD-1 was measured byFACS and ELISA assay. FIG. 16 shows the binding of 1.103.11-v2 hAbs indifferent buffers to PD-1 expressing CHO cell, and the binding was alsotested under the same condition as that of FACS assay as describedabove, except that the antibody was either in formulation buffer or in1×PBS (pH 7.4) and the CHO-S cells expressing human PD-1 weretransferred in to 96-well U-bottom plates (BD) at a density of 2×10⁵cells/ml. The result was comparable to that of 1.103.11 hAb.“1.103.11-v2 hAb in buffer” refers to the antibody in the formulationbuffer, and “1.103.11-v2 hAb in PBS” refers to antibody in the 1×PBS, pH7.4. Antibodies in both solutions bound to cell surface PD-1 on the CHOcell and there was no significant difference in affinity to human PD-1between the two conditions (for 1×PBS the EC50 was about 2.52 nmol/L,and for formulation buffer it was about 3.12 nmol/L).

FIG. 15 shows the binding of antibody 1.103.11-v2 hAbs to PD-1 proteinin different solutions measured by ELISA. Following the same ELISAprotocol as described above, the incubation time for 1.103.11-v2 hAb was2 h, and the incubation time for the secondary antibody goat anti-humanIgG FcHRP (1:5000, Abcam) was 1 h. “1.103.11-v2 hAb in buffer” refers tothe antibody in the formulation buffer, and “1.103.11-v2 hAb in PBS”refers to antibody in the 1×PBS (pH 7.4). Binding affinity to human PD-1was demonstrated under both conditions.

CHO cells expressing human PD-1 were incubated with differentconcentrations of the antibodies against PD-1, then the mouse Fc-taggedhuman PD-L1 was added to the cells. The binding of human PD-L1 to PD-1expressing cell was detected by using FITC-conjugated goat anti-mouseIgG, followed by the FACS analysis. As shown in FIG. 4 , antibodiesagainst PD-1 blocked the binding of PD-L1 to PD-1 transfected CHO cells.The 1.103.11-v2 hAb was also tested for blockade of PD-L1 binding toPD-1 transfected CHO cells, and the result was comparable to that of1.103.11 hAb.

3.2 Full kinetic binding affinity tested by surface Plasmon resonance(SPR): Antibodies were characterized for affinity and binding kineticsto PD-1 by SPR assay using ProteOn XPR36 (Bio-Rad). Protein A protein(Sigma) was immobilized to a GLM sensor chip (Bio-Rad) through aminecoupling. Purified antibodies were flowed over the sensor chip andcaptured by the Protein A. The chip was rotated 90° and washed withrunning buffer (1×PBS/0.01% Tween20, Bio-Rad) until the baseline isstable. Five concentrations of human PD-1 and running buffer were flowedagainst the antibody flow cell at a flow rate of 100 μL/min for anassociation phase of 240 s, followed by 600 s dissociation. The chip wasregenerated with pH 1.7 H₃PO₄ after each run. The association anddissociation curve was fit to a 1:1 Langmiur binding model using ProteOnsoftware.

As shown in FIG. 7 , using surface plasmon resonance, the affinities ofantibodies against PD-1 for recombinant human PD-1 were from 3.76E-9 to1.76E-10 mol/L. The affinity of 1.103.11-v2 hAb is expected to becomparable to that of 1.103.11 hAb.

3.3 Orthologue (cross-species) and homologue (cross-families) screen:

3.3.1 Cross-reactivity to cynomolgus PD-1 and murine PD-1:Cross-reactivity was measured by ELISA. Plates (Nunc) were coated withcynomolgus PD-1 (Sino Biological) and murine PD-1 (Sino biological) at 1μg/ml overnight at 4° C. After blocking and washing, 1 μg/ml antibodieswere added to the plates and incubated at room temperature for 1 h. Theplates were then washed and subsequently incubated with secondaryantibody goat anti rat IgG1 HRP (Bethyl) and goat anti rat IgG2b HRP(Bethyl) for 45 min. After washing, TMB substrate was added and theinteraction was stopped by 2M HCl. The absorbance at 450 nm was readusing a microplate reader (Molecular Device).

The result of cross-species experiment demonstrates that antibodiesagainst PD-1 bind to cynomolgus monkey PD-1 but not bind to murine PD-1(FIG. 6 ). The 1.103.11-v2 hAb in the same experiment is expected tohave comparable result to that of 1.103.11 hAb.

3.3.2 Cross-reactivity to PD-1 family members CD28, CTLA4 and ICOS: toexamine the cross-family binding activity of the fully human antibodies,cells lines that express PD-1, CD28, CTLA4 or ICOS were stained with theantibodies, followed by 2′ antibody staining with FITC conjugated goatanti-human IgG Fc. PD-1 expressing cells were used as positive control.Corresponding parental cell lines were used as negative controls. Thestained cells were analyzed by using a BD Biosciences FACSCanto II andFlowJo Version software.

FIG. 5 shows that CHO cells transfected with PD-1, CD28 and 293Ftransfected with CTLA4 were stained with antibodies against PD-1 andanalyzed by FACS. The result demonstrates PD-1 antibodies bindspecifically to PD-1, but not to CD28 and CTLA4 of PD-1 family. The1.103.11-v2 hAb in the same experiment is expected to have comparableresult to that of 1.103.11 hAb.

3.4 Epitope binning test:

3.4.1 The binding epitope of PD-1 antibodies was binned againstbenchmark antibody A and B by SPR assay using ProteOn XPR36 (Bio-Rad.Benchmark antibodies A and B were immobilized on GLC sensor chip(Bio-Rad) through amine coupling. Human PD-1 solution was flowed overthe antibody immobilized channels and captured by the benchmarkantibodies. The chip was then rotated 90° and washed with running bufferuntil the baseline is stable. Selected antibodies were flowed over thesensor chip.

3.4.2 The binding epitope of PD-1 antibodies was binned againstbenchmark antibody A and B by FACS. CHO cells expressing human PD-1 atthe cell surface were incubated with benchmark antibody A or B atconcentration of 10 μg/ml for 1 hour. The cells were washed and the PD-1antibodies of the disclosure were added and incubated for 1 hour. Thesecond antibody anti-rat IgG-FITC were added and incubated for 1 hour at4° C. The cells were then washed once and resuspended in 1×PBS/1% BSA,and analyzed by flow cytometery (BD).

The results of SPR assay and FACS for the binning test showed that theepitope on human PD-1 bound by the fully human PD-1 antibodies (i.e.1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb) wasdifferent from the existing PD-1 antibodies (i.e. benchmark antibody Aand B). The 1.103.11-v2 hAb in the same experiment is expected to havecomparable result to that of 1.103.11 hAb.

3.5 In vitro function of PD-1 antibodies tested by cell-based assays:

3.5.1 Effects of human PD-1 antibodies on T cells proliferation. Anallogeneic response was used to test the effects of PD-1 antibodies on Tlymphocytes proliferation. Primary dendritic cell (DC)-stimulated MLRwas conducted in 96-well, U-bottom tissue culture plates in 200 μl ofRPMI 1640 containing 10% FCS and antibiotics. DCs were mixed with 1×10⁵allogeneic total CD4±T cells at a ratio between 1:10 and 1:100 DC:Tcells. Cultures were also conducted in the presence or the absence ofneutralizing mAbs: human PD-1 antibodies and benchmark antibody A and Band used at 10 μg/ml. Assays were incubated for 5 days, and during thelast 16 h [³H]thymidine was added at 1 uCi/well. [³H]thymidineincorporation was measured by scintillation counting, and proliferativeresponses were expressed as the mean [³H]thymidine incorporation (countsper minute) of triplicate wells. Counts due to DCs alone were routinely<1000 cpm. Results shown are representative examples of a minimum offive experiments performed.

Human dendritic cells (DC) and CD4⁺T, CD8⁺T and total cells used inabove allo-MLR were generated from the PBMC as following procedures:Human monocytes were purified from PBMC by negative selection usinghuman monocyte enrichment cocktail kit according to the instructions ofthe manufacturer (StemCell Meylan). Briefly, PBMC were isolated fromblood of healthy donor using a Ficoll-Paque gradient. Cells were washedtwice with PBS, then resuspended at 1×10⁸ cells/ml in isolation buffer,and incubated with the monocyte enrichment Ab mixture at 4° C. for 30min. The cells were washed and subsequently incubated with magneticcolloid at 4° C. for 30 min. Unlabeled monocytes passed through the MACScolumn and were collected. To generate iDCs, monocytes were cultured inRPMI 1640 medium containing 10% FCS and antibiotics with GM-CSF(PeproTech, Rocky Hill, NJ; 800 U/ml) and IL-4 (PeproTech; 500 U/ml) atconcentration of 2×10⁶ cells/ml. Half the medium was replaced everyother day with GM-CSF- and IL-4-containing medium. Mature DCs weregenerated by stimulating iDCs with LPS (026: B6; Sigma-Aldrich, St.Louis, MO; 1 μg/ml) on day 5 for an additional 24 h. CD4⁺T, CD8⁺T andtotal T cells, were purified by negative selection by incubating PBMCwith human CD4⁺T, CD8⁺T and total T cell enrichment mixture and magneticcolloid according to the manufacturer's instructions (Stemsep).

Human CD4⁺ T Cells were stimulated with allogenenic DCs in the presenceor absence of PD-1 antibodies 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb,1.139.15 hAb, and 1.153.7 hAb. The proliferation of CD4⁺ T cells wereassessed by [³H] thymidine incorporation. FIG. 10 showed that 1.7.3 hAb,1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb enhancedconcentration dependent T cell proliferation. The 1.103.11-v2 hAb in thesame experiment is expected to have comparable result to that of1.103.11 hAb.

3.5.2 Effects of human PD-1 antibodies on cytokine IFNγ secretion invitro: to directly assess the effect of human PD-1 antibodies blockadeon cytokine IFNγ production, we performed experiments on IFNγ productionin allo-MLR. Briefly, human CD4⁺T cells were purified from PBMC bynegative selection with CD4⁺T cell enrichment cocktail kit according tothe instruction of the manufacturer. Immature DCs were generated frommonocytes by cultured in GM-CSF and IL-4 for 5 days and mature DCs weredifferentiated by stimulation with LPS at 1 μg/ml for overnight. CD4±Tcells were mixture with iDC/mDc at a ratio between 10:1 and 100:1 T:DCratio. The cultures were conducted in the presence or absence of humanPD-1 antibodies and benchmark antibodies. After 5 days, the supernatantsfrom each culture were harvested for cytokine IFNγ measurement. Thelevel of IFNγ in supernatants was measured by ELISA assay. Briefly, CoatMaxisorp plates with anti-human IFN-gamma mAb diluted in coating buffer(0.75 μg/ml; i.e. a 1/1360 dilution), 50 μl/well (i.e. for a full96-well plate add 3.7 μl of antibody to 5 ml of coating buffer) andincubated overnight at 4° C. Block spare protein binding capacity byadding 200 μl/well of blocking buffer for 2 hours. Prepare dilutions ofrecombinant IFN-gamma to act as standards, two-fold dilutions from 8000pg/ml down to 125 pg/ml, diluted in complete medium, plus completemedium alone. Wash plates and add standards and test supernatants (100μl/well), incubate for 2-4 hours. The biotinylated anti-IFN-gamma mAb (1/1333) in blocking buffer was added followed by adding Extra-avidinPeroxidase. The reaction was developed by adding TMB substrate andstopped with 2M HCl. Measure absorbance at 450 nm.

FIG. 9 shows that human CD4⁺ T Cells were stimulated with allogenenicDCs in the presence or absence of antibodies 1.7.3 hAb, 1.49.9 hAb,1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb. The level of IFNγ wasmeasured by ELISA. The result showed the fully human PD-1 antibodiesincreased IFNγ secretion in a dose manner. The 1.103.11-v2 hAb in thesame experiment is expected to have comparable result to that of1.103.11 hAb.

3.5.3 Effects of human PD-1 antibodies on interleukin 2 (IL-2)production in vitro: CD4⁺ T cells were mixture with iDC/mDc at a ratiobetween 10:1 and 100:1 T:DC ratio. The cultures were conducted in thepresence or absence of human PD-1 antibodies and benchmark antibodies.After 5 days, the supernatants from each culture were harvested forcytokine measurement. The level of IL-2 in supernatants was measured byELISA assay.

FIG. 8 shows that human CD4⁺ T Cells were stimulated with allogenenicDCs in the presence or absence of lead antibodies or control Ab. Thelevel of IL-2 was measured by ELISA. The results showed antibodiesagainst PD-1 increased IL-2 secretion in a dose-dependent manner. The1.103.11-v2 hAb in the same experiment is expected to have comparableresult to that of 1.103.11 hAb.

3.5.4 Effect of human PD-1 antibodies on cell proliferation and cytokineproduction by autologous antigen specific immune response: in thisassay, the T cells and DCs were from a same donor. Briefly, CD4⁺T cellwere purified from PBMC and cultured in the presence of CMV pp65 peptideand low dose of IL2 (20 U/ml), at the meantime, DCs were generated byculturing monocytes from the same donor's PBMC in GM-CSF and IL-4. After5 days, the CMV pp65 peptide treated CD4⁺T cells were co-cultured withDCs pulsed with pp65 peptide in the absence or presence of human PD-1antibodies and benchmark antibodies (as control). On day 5, 100 μl ofsupernatants were taken from each of cultures for cytokine IFNγ and IL-2measurement. The level of IFNγ and IL-2 production was detected by ELISAassay. The proliferation of specific T cells to CMVpp65 peptide-pulsedDCs were assessed by [³H]thymidine incorporation.

As shown in FIG. 11 , PD-1 antibodies enhanced concentration dependentCMV⁺-CD4⁺ T cell proliferation stimulated with CMV pp65 peptide-loadedautologous DC. The 1.103.11-v2 hAb in the same experiment is expected tohave comparable result to that of 1.103.11 hAb.

3.5.5 Effect of human PD-1 antibodies on regulatory T cell (Tregs)suppressive function: Tregs, a subpopulation of T cells, are a keyimmune modulator and play key roles in maintaining self-tolerance.CD4⁺CD25⁺ regulatory T cell are associated with tumors because increasednumbers of Tregs were found in patients with multiple cancers and isassociated with a poorer prognosis. To directly assess the effect ofhuman PD-1 antibodies on immune suppressive response, we performedexperiment on Tregs. CD4⁺CD25⁺ and CD4⁺CD25⁻T cells were separated usingspecific anti-CD25 microbeads (Miltenyi Biotec, Auburn, CA) and positiveor negative selection, respectively. Initially, CD4⁺T cells werepurified by negative selection by incubating PBMC with human CD4⁺T cellenrichment mixture and magnetic colloid according to the manufacturer'sinstructions (Stemsep). CD4⁺T cells were then resuspended in MACSbuffer, incubated with CD25⁺microbeads on ice for 30 min, washed, andloaded on the column. CD4⁺CD25⁻ T cells, which did not bind to thecolumn, were collected from the flow-through and washed before use.CD4⁺CD25⁺T cells were subsequently retrieved from the column and washedbefore use. Tregs were cultured with CD4⁺CD25⁻T cells and DCs (Treg:Teff1:1 ratio) in the presence or absence of human PD-1 antibodies at aconcentration of 10 μg/ml. Either no antibody or isotype antibody wasused as negative control. The supernatants from the cultures were takenon day 5 for cytokines detection by ELISA and the cell proliferation wasmeasured by adding [³H]thymidine at a concentration of 1 uCi/well andincubated for further 18 hours. [³H]thymidine incorporation was measuredby scintillation counting. As shown in FIG. 12 , the PD-1 antibodiesabrogated Treg's suppressive function and restored responding T cellproliferation and IFNγ secretion. The 1.103.11-v2 hAb in the sameexperiment is expected to have comparable result to that of 1.103.11hAb.

3.6 ADCC/CDC assay: to minimize the undesired toxicity on healthy PD-1⁺cells, the selected anti-PD-1 fully human antibodies were confirmed tohave no ADCC and CDC function.

3.6.1 ADCC: Activated T cells expressing high levels of cell surfacePD-1 were used as target cells and were pre-incubated with variousconcentrations of fully human antibodies in 96-well plates for 30 min,then IL-2-activated PBMCs (used as a source of natural killer (NK)cells, i.e. the effector cells) were added at the effector/target ratioof 50:1. The plates were incubated for 6 hours at 37° C. in a 5% CO₂incubator. Target cell lysis was determined by cytotoxicity detectionkit (Roche). Optical density was measured by Molecular DevicesSpectraMax M5e Plate Reader. Results showed that, the tested fully humanantibodies against PD-1 did not mediate ADCC (FIG. 13 ). The 1.103.11-v2hAb in the same experiment is expected to have comparable result to thatof 1.103.11 hAb.

3.6.2 CDC: target cells (activated T cell), diluted human serumcomplement (Quidel-A112) and various concentrations of fully human PD-1antibodies were mixed in a 96-well plate. The plate was incubated for 4h at 37° C. in a 5% CO₂ incubator. Target cell lysis was determined byCellTiter glo (Promega-G7573). Rituxan (Roche) and human B lymphoma cellline Raji (CD20 positive) were used as positive control. The data showedthat PD-1 antibodies did not mediated CDC (FIG. 14 ). The 1.103.11-v2hAb in the same experiment is expected to have comparable result to thatof 1.103.11 hAb.

Example 4: Epitope Mapping of the Fully Human Antibody

To determine the epitope difference between the present antibody1.103.11 hAb provided herein and Keytruda, a known hPD-1 antibody,alanine scanning experiments on hPD-1 and the effect evaluation toantibody binding were conducted using 1.103.11 hAb, Keytruda and11.148.10 (a control hPD-1 antibody which binds to an epitope which doesnot overlap with that of 1.103.11 hAb or that of Keytruda).

Alanine residues on hPD-1 were mutated to glycine codons, and all otherresidues were mutated to alanine codons. For each residue of the hPD-1extracellular domain (ECD), point amino acid substitutions were madeusing two sequential PCR steps. A pcDNA3.3-hPD-1_ECD.His plasmid thatencodes ECD of human PD-1 and a C-terminal His-tag was used as template,and a set of mutagenic primer was used for first step PCR using theQuikChange lightning multi-site-directed mutagenesis kit (Agilenttechnologies, Palo Alto, CA). Dpn I endonuclease was used to digest theparental template after mutant strand synthesis reaction. In thesecond-step PCR, linear DNA expression cassette which composed of a CMVpromoter, an extracellular domain (ECD) of PD-1, a His-tag and a herpessimplex virus thymidine kinase (TK) polyadenylation was amplified andtransiently expressed in HEK293F cells (Life Technologies, Gaithersburg,MD).

Monoclonal antibodies 1.103.11 hAb, keytruda and 11.148.10 hAb werecoated in plates for ELISA binding assay. After interacting with thesupernatant that contains quantified PD-1 mutant, HRP conjugatedanti-His antibody (Rockland, Cat #200-303-382) was added as detectionantibody. Absorbance was normalized according to the average of controlmutants. After setting an additional cutoff to t4he binding fold change(<0.55), the final determined epitope residues were identified.

Top 30 point-substituted hPD-1 mutants that significantly reducedantibody binding were shown in Table 2. Checking the positions of allthese residues on the hPD-1 crystal structures (PDB code 3RRQ and 4ZQK)revealed that some amino acids (e.g. Val144, Leu142, Val110, Met108,Cys123 etc.) were fully buried in the protein, and were unlikely todirectly contact any antibodies. The observed binding reductions mostprobably resulted from the instability or even collapse of hPD-1structure after alanine substitutions. To avoid misinterpreting thesedata as epitope hot spots, we took advantage of a control antibody11.148.10 hAb, which binds to a quite different location on antigen, butis expected to respond to the collapse of the hPD-1 structure if itindeed happens. Mutants that affect both antibodies were treated asfalse hot spots and were removed from the list. After setting anadditional cutoff to the binding fold change (<0.55), the finaldetermined epitope residues were listed in Table 3. They are 9 positionsto 1.103.11 hAb and 5 positions to Keytruda, and 10 residues to thecontrol antibody 11.148.10 hAb.

TABLE 2 The effect of PD-1 point mutations on antibody binding 1.103.11hAb Keytruda 11.148.10 hAb PD-1 fold PD-1 fold PD-1 fold #Residue changeª SD #Residue change ª SD #Residue change ^(a) SD V 144 0.22 0.00 P 890.18 0.02 V 144 0.03 0.01 A 129 0.22 0.00 D 85 0.38 0.01 F 56 0.06 0.02D 85 0.24 0.01 V 144 0.40 0.01 L 142 0.09 0.00 P 83 0.30 0.01 R 94 0.460.04 D 48 0.21 0.01 L 128 0.32 0.01 F 106 0.47 0.05 R 143 0.26 0.01 V 640.32 0.01 K 78 0.48 0.00 C 123 0.27 0.01 Q 133 0.37 0.03 P 83 0.50 0.01F 106 0.29 0.04 P 130 0.41 0.00 D 92 0.50 0.02 V 44 0.34 0.01 F 106 0.410.02 P 39 0.54 0.00 L 41 0.35 0.00 K 131 0.43 0.01 A 81 0.57 0.01 A 500.35 0.02 L 142 0.44 0.00 C 123 0.57 0.01 F 95 0.37 0.01 C 123 0.46 0.00N 66 0.57 0.03 V 43 0.37 0.01 A 132 0.53 0.01 L 142 0.59 0.01 V 110 0.410.01 P 39 0.55 0.02 F 82 0.61 0.03 M 108 0.43 0.11 M 108 0.56 0.00 F 950.61 0.04 R 94 0.46 0.12 F 52 0.59 0.00 F 52 0.63 0.01 C 93 0.48 0.03 K135 0.62 0.01 M 108 0.64 0.06 R 86 0.49 0.01 S 137 0.62 0.01 L 128 0.680.01 D 117 0.49 0.12 F 95 0.63 0.02 I 126 0.72 0.01 A 113 0.51 0.01 I126 0.64 0.01 A 113 0.72 0.01 T 45 0.51 0.03 F 82 0.65 0.01 V 110 0.730.04 L 42 0.53 0.01 I 134 0.69 0.01 G 47 0.73 0.01 A 40 0.54 0.00 R 940.70 0.01 D 117 0.73 0.07 P 39 0.55 0.00 A 50 0.73 0.01 N 49 0.73 0.00 G90 0.56 0.08 D 117 0.73 0.01 S 87 0.74 0.06 N 49 0.58 0.01 A 113 0.730.02 L 42 0.76 0.01 S 137 0.58 0.02 N 49 0.73 0.01 N 102 0.76 0.01 Y 680.58 0.03 L 65 0.75 0.01 W 67 0.81 0.01 W 67 0.60 0.03 W 67 0.76 0.01 P101 0.81 0.04 F 52 0.60 0.05 G 47 0.77 0.00 A 80 0.82 0.01 R 69 0.610.05 Bold: amino acids overlapped with control 11.148.10 hAb forstructure maintaining which were excluded from the hot spots list. ^(a)Fold change in binding is relative to the binding of several silentalanine substitutions.

TABLE 3 Identification of potential epitopes PD-1 to residue PD-1 toresidue PD-1 to residue 1.103.11 hAb location Keytruda location11.148.10 hAb location V 64 C K 78 C′ L 41 A′ P 83 C′ P 83 C′ V 43 A′ D85 C″ D 85 C″ V 44 A′ L 128 FG P 89 C″ T 45 A′ A 129 FG D 92 C″D D 48A′B P 130 FG A 50 B K 131 G F 56 B A 132 G R 86 Q 133 G C 93 C″′D R 143G Cutoff: fold change < 0.55 * C, C′, C″, F, G, A′ represent the peptidestrands in the crystal structure of the hPD-1 as shown in FIG. 17. TheC″ strand observed on mPD-1 doesn′t exist on hPD-1 structure. Thisβ-sheet is replaced by a structureless loop on hPD-1. We still use C″ tolabel this region, just for the purpose of easier comparison to mPD-1.

Comparing the epitope residues of 1.103.11 hAb and Keytruda in Table 3only revealed two overlapped hot spot residues. The rest looked quitediverse, which indicated that two antibodies might have adopted verydifferent mechanisms in terms of hPD-1 binding and hPD-L1 blocking.Reading the residue IDs in Table 3 is not straightforward to interpretthe mechanisms. All data in Table 3, as well as the hPD-L1 binding site,were therefore mapped on the crystal structure of hPD-1 to make a bettervisualization and comparison. (FIG. 17 ).

As shown in FIG. 17 , the hot-spot residues in charge of the hPD-L1binding all gathered in the middle of C, F and G strands (FIG. 17A). Twoinvestigated antibodies 1.103.11 hAb and Keytruda, although both arefunctional in binding hPD-1 and blocking hPD-L1, have obviouslydifferent epitopes (FIG. 17B for 1.103.11 hAb, 17C for Keytruda). Theepitope of Keytruda were mainly contributed by the residues on the C′Dloop (corresponding to the C″ strand on mPD-1), which didn't intersectthe PD-L1 binding site at all. This suggested the hPD-L1 blockingfunction of Keytruda relied more on its steric hindrance effectsprovided by the size of the antibody. In contrast, the epitope of ourlead antibody 1.103.11 hAb was composed of hot spots distributed acrossmultiple locations, and have direct overlap with the hPD-L1 binding site(FIG. 17A, 17B). The present 1.103.11 hAb blocked hPD-L1 by means ofbeing more competitive than hPD-L1 in reacting to their common bindingsite. 1.103.11 hAb is therefore expected to be more functional indownstream developments.

Antibody 11.148.10 hAb, however, binds to a completely differentlocation (FIG. 17D) from two functional antibodies, which confirmeditself a good control antibody to monitor the function of hPD-1 inperforming alanine substitutions.

While the disclosure has been particularly shown and described withreference to specific embodiments (some of which are preferredembodiments), it should be understood by those having skill in the artthat various changes in form and detail may be made therein withoutdeparting from the spirit and scope of the present disclosure asdisclosed herein.

1-34. (canceled)
 35. A monoclonal anti-programmed cell death 1 (PD-1)antibody comprising a heavy chain variable region comprising SEQ ID NO:53; a light chain variable region comprising SEQ ID NO: 67; and a humanconstant region of IgG4 isotype.
 36. The antibody of claim 35, whereinthe antibody is a fully human monoclonal antibody.
 37. A pharmaceuticalcomposition comprising the antibody of claim
 35. 38. An isolatedpolynucleotide encoding the antibody of claim
 35. 39. A vectorcomprising the isolated polynucleotide of claim
 38. 40. A host cellcomprising the vector of claim
 39. 41. A method of treating a conditionassociated with PD-1 in an individual, comprising: administering atherapeutically effective amount of the antibody of claim
 35. 42. Themethod of claim 41, wherein the condition is cancer or chronic viralinfection.